Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily

Phosphomannose IsomerasesPhosphomannose isomerases (PMI) (EC 5.3.1.8 ) are enzymes that catalyze the interconversion of mannose-6-phosphate and fructose-6-phosphate. The subclass most relevant to this review is that of the type II enzymes (139, 227), known to be involved in a variety of microbial pathways including capsular polysaccharide biosynthesis and d-mannose metabolism. Such enzymes, which contain the two-motif cupin signature separated by 15 aa, exist either as a single-function protein of about 120 to 150 aa or as the C-terminal domain of a bifunctional enzyme (ca. 480 aa) with both PMI and GDP-mannose pyrophosphorylase (GMP) (EC 2.7.7.22 ) activity. An example of the latter type of protein, and one of particular practical importance, is the 56-kDa bifunctional enzyme encoded byalgA (179, 196, 259), which catalyzes the first and third steps in the biosynthesis of alginate, PMI catalyzing the first step (152). This compound is composed of 1,4-linked α-l-guluronic acid and β-d-mannuronic acid and is of great economic importance, although for commercial production it is usually extracted from marine seaweeds rather than from bacteria (237). Alginate also has medical significance because of its production by Pseudomonas aeruginosa during the conversion of this bacterium to a mucoid form (256). This conversion is induced by several conditions: starvation, the presence of metabolic inhibitors, or, most importantly, growth of the bacteria in the lungs of cystic fibrosis patients. Indeed, mortality in such patients is usually associated with the inability of antibiotics to penetrate the bacterial biofilm and to the fact that the alginate protects the bacteria from the host immune responses (136). Similarly, alginate is a major component of metabolically dormant cysts in the aerobic nonsymbiotic soil bacterium Azotobacter vinelandii, where it may account for up to 70% of the intine (inner layer of wall) and 40% of the exine (outer layer of wall) carbohydrates. This coating is believed to protect the cell from desiccation and other stresses, and indeed its production in the lungs of cystic fibrosis patients may be linked to the need for the bacterial cells to protect themselves from the dehydrating environment.

The equivalent bifunctional enzyme in Escherichia coli is ManC (gi‖3435180), part of the biosynthetic pathway for GDP-l-fucose and GDP-perosamine, components of the O-antigen gene cluster (140, 280, 283, 301). Other related bacterial enzymes include those encoded by noeJ fromRhizobium (81) and aceF, which is part of the acetan biosynthetic pathway in Acetobacter xylinus(102). There are also related genes in the archaeal speciesPyrococcus horikoshii (gi‖3257338), Methanobacterium thermoautotrophicum (gi‖2622642), and Archaeoglobus fulgidus (gi‖2649495).

Because of its importance in the synthesis of bacterial and fungal cell walls, PMI inhibition is a target for drug discovery (32). Although there is limited information on the structure of the active site, in the context of the conserved histidines in the two cupin motifs it is pertinent to note recent evidence (220) for the existence of a His residue in this site in a PMI from Xanthomonas campestris; this particular PMI is considered to be a metalloenzyme and is activated by zinc.

Polyketide Synthases (Putative Cyclases)The polyketide pathway (115, 125, 194, 235) accounts for the biosynthesis of many of the thousands of known secondary metabolites, including antibiotics and pigments. Among these products is curamycin (26), an antibiotic produced byStreptomyces curacoi and based on a polyketide skeleton consisting of a modified orsellinic acid—an unreduced version of 6-methylsalicylic acid and the simplest of all aromatic polyketides. It was found (25) that the gene cluster responsible for the synthesis of this antibiotic was very similar to the S. coelicolor whiE gene cluster responsible for the synthesis of a grey spore pigment produced shortly before sporulation in the aerial mycelium (51), and subsequent studies (36) demonstrated the widespread occurrence of gene clusters very similar towhiE among other Streptomyces spp. Of specific interest to this review is the sequence of the homologous group of genes represented by curC (S. curacoi),whiE ORFII (S. coelicolor) (8),sch ORFB (S. halstedii), and tcmJ(S. glaucescens) (33). The exact biochemical function of these gene products remains unknown, although it is suggested to be a cyclase (148, 318). Sequence analysis reveals the two conserved cupin motifs, separated by a distance of 15 residues, within a total protein size of approximately 150 aa. It has been suggested recently (12) that use of the CurC sequence is the most efficient means of identifying other members of the cupin family in a PSI-BLAST search (7).

Recent analysis (69) has extended the number of members in this particular cupin subfamily to include several other close relatives, such as the sequence gi‖2635101 (YrkC) from B. subtilis and the 140-aa Pep1 sequence (gi‖1572541) encoded by gene tnpA of the cryptic transposon Tn4321 within the broad-host-range IncPβ plasmid R751 of Enterobacter aerogenes (267, 291). The notes accompanying the Pep1 database submission recognized a “possible polyketide cyclase on basis of weak similarity to TcmJ of S. glaucescens” (E value 8.5). However, it is most similar (E value 8e-08) to a 97-aa sequence encoded by nucleotides 180 to 467 of a contig (gnl‖Stanford_382‖smelil_423025B02.xl) fromSinorhizobium meliloti.

It was assumed previously that the smallest of all cupins is the 77-aa “membrane-spanning protein” gi‖1017816 from Streptomyces coelicolor (181, 182). However, the start codon for this sequence has been reassigned, and it is now considered to encode a 115-aa protein (gi‖5457273) that is most similar to a 79-aa polypeptide encoded by nucleotides 243111 to 243347 from contig 7 ofStreptococcus pyogenes.

DioxygenasesSeveral types of dioxygenase enzymes are probable members of the cupin superfamily. They can be divided into two categories, those with a single domain and those with two domains (bicupins); within each subcategory the individual members can be recognized on the basis of a characteristic inter-motif spacing.

3-Hydroxyanthranilate 3,4-dioxygenase (3-HAO) (EC 1.13.11.6 ), with an intermotif spacing of 19 or 23 aa, is a eukaryotic enzyme that cleaves the aromatic ring of 3-hydroxyanthranilic acid to produce 2-amino-3-carboxymuconic semialdehyde, an intermediate in the synthesis of the excitotoxin quinolinic acid (21); this compound kills neurons by activation of N-methyl-d-aspartate receptors, and inhibition of 3-HAO is therefore a pharmaceutical target (35). The enzyme is well characterized in mammals (210) and is part of the kynurenine pathway for the catabolism of tryptophan. Recently, the yeast gene YJR025chas been shown (164) to encode a 3-HOA (gi‖1353060) homologous to the human equivalent (190) and has been renamed BNA1 (biosynthesis of nicotinic acid). A very similar polypeptide (E value 5e-64) is encoded by part of a contig (gnl‖Stanford_5476‖C.albicans_Con4-2428) from Candida albicans. Alignment of these 3-HOA sequences shows a notable difference between the Saccharomyces sequence and the other sequences, in that the former protein has an intermotif spacing of 23 residues compared with 19 for the other sequences. This insertion of 4 aa occurs in the loop between the E and F strands of the barrel.

In common with most other dioxygenase enzymes, 3-HAO requires nonheme iron as a cofactor. However, in contrast to the multimeric composition of the related, two-domain dioxygenases described below, this enzyme seems to be monomeric.

Cysteine dioxygenase (CDO) (EC 1.13.11.20 ), with an intermotif spacing of 28 residues, is a key enzyme of cysteine metabolism and catalyzes the production of cysteine sulfinate. The rat (296), human (232), and Caenorhabditis elegans genes have been well characterized, with the closest bacterial relatives of these eukaryotic sequences being those from B. subtilis(gi‖2635598), Streptomyces coelicolor (gi‖2687337), andMycobacterium tuberculosis (gi‖2896702). This enzyme is known to be monomeric, with one atom of iron per molecule (312); its activity is strongly reduced by chelators of Cu⁺ and Fe²⁺ (247).

SpherulinsThe life cycle of the simple slime mold Physarum polycephalum involves a transition between two vegetative states, the amoeba and the plasmodium. Amoebae are the uninucleate haploid cells, which under some conditions will fuse and differentiate into a giant multinucleate diploid plasmodium. When these latter cells are grown in liquid medium, they fragment into microplasmodia, which are capable of withstanding adverse conditions by encystment. This transition into hard-walled oligonucleated spherules is termed spherulation, and it is induced by starvation (or high concentrations of some carbohydrates), cooling, dehydration, acidic pH, and/or sublethal concentrations of heavy metals (47, 144).

As part of a molecular study of this phase transition, it was shown first that the major changes in protein synthesis take place 24 h after the beginning of starvation-induced spherulation (31) and that the four most abundant spherulation-specific RNAs accounted for more than 10% of all mRNAs present after this period (30); these mRNAs were not present in encysting amoebae or in sporulating plasmodia. Differential hybridization of a cDNA library was used subsequently to isolate full-length clones (29), of which two were found to be 76% similar and encoded proteins named spherulins 1a and 1b (81% identical). These proteins possess a potential signal peptide and an N-glycosylation site and were therefore presumed to be cell-wall glycoproteins. It was discovered subsequently (171) that there is 44% similarity at the amino acid level between spherulin 1b and the wheat germin GF-2.8; this value increases to 60% for the central core sequence, the region that contains the conserved PH(I/T)HPRATEI decapeptide designated the germin box. They can thus be considered cupins, with an intermotif spacing of 21 aa.

An interesting addition to the discussion on the evolutionary origin of the spherulin genes is provided by an analysis of intron position (20) in a series of related cupins. The discovery that the C-terminal domain of several seed storage proteins (e.g., those ofWelwitschia mirabilis and Gingko biloba) shared an intron position with the spherulins (although shifted by 2 bp inP. polycephalum) provided strong support for the concept that these proteins have a common ancestor.

To date, no biochemical function has been assigned to these spherulins, although they do not seem to have any OXO activity (173). However, it is relevant to consider their possible function(s) in the specific context of what is known about the conditions pertaining during spherulation and also in the general context of the link between cupins and stress responses in prokaryotes and eukaryotes. In particular, it is interesting to note the link between oxidative stress and spherulation. The initial circumstantial evidence for such a link came from the observation (2) that the herbicide paraquat, a compound that generates free radicals, accelerated spherulation and also increased the specific activity of the manganese isoform of superoxide dismutase (3). It was also found that during the spherulation process in salts-only starvation medium, superoxide dismutase activity increased 46-fold, along with an increase in the concentrations of H₂O₂ and organic peroxide (2); none of these changes occurred in nondifferentiating cultures.

Germin and Germin-Like Proteins from Higher PlantsWheat germin (which is an OXO), is the best characterized of all the cupin proteins in terms of its biochemistry, function, and patterns of expression (45); it is therefore particularly relevant to consider these various features in some detail. The first evidence for such an enzyme that converts oxalic acid and dioxygen to carbon dioxide and hydrogen peroxide came from studies of powdered wheat grains in 1912 (320), although it was more than 80 years later that the identity and sequence of this enzyme were confirmed (173). In the meantime, there had been two parallel and unrelated types of research concerning this particular protein. The first of these concerned an important medical application of considerable commercial significance, namely, the use of barley OXO (98% identical to wheat germin) in kits to assay levels of oxalate in blood plasma and urine. Some of these kits (e.g., the Sigma kit) utilize an enzyme isolated from barley roots, and although they are quick and easy to use, there is a continuous effort to improve the accuracy and efficiency of the assay (175, 191, 228). Such efforts will benefit from recently obtained data regarding fundamental biochemical and structural analysis of the barley enzyme itself (161, 162, 238, 310) and from the finding (173) that the extremely well characterized wheat germin is also an OXO. This discovery was the culmination of the second important research track, one which started in the early 1980s, during which the GF-2.8 germin (gi‖121129) was found to be an apoplastic, multimeric (310), glycosylated (135) enzyme with extreme resistance to heat and to chemical degradation by protease or hydrogen peroxide. These unusual properties have recently been explained by the realization that wheat germin and its relatives from barley and other cereals (206) are members of the cupin family and that their resistance to extremes of environment is likely to be a function of their structural similarity to other desiccation-tolerant proteins including 7S and 11S seed storage proteins; the resistance of the protein to H₂O₂ is of course linked to its enzymatic generation of this compound.

Germin-like proteins (GLPs) have a maximum ca. 90% sequence identity (e.g., gi‖1772596) to wheat germin, although the average level of identity is closer to 50%. There is almost complete identity in the conserved cupin core, in which the intermotif spacing is 20 to 23 aa. Since the discovery of the first GLP in a higher plant (127), there has been a rapid expansion in the number of gene sequences identified, such that the latest estimates give a total of 21 sequences in Arabidopsis thaliana, the best-characterized plant genome to date (46; J. M. Dunwell, unpublished data). However, no function has yet been assigned to any of these sequences, with the single exception of aPinus caribaea GLP, which does have OXO activity (212). In addition to the identification of GLP genes in analyses of various plant genomes, expression of certain GLPs in plants, including liverworts (gi‖4718551) and mosses (gi‖6042701, gi‖6102532), is associated with a range of specific developmental states but more particularly with specific biotic and abiotic stresses, as detailed below.

Auxin-Binding ProteinsAuxin-binding proteins (ABPs) (intermotif spacing of 24 aa) are dimeric, glycosylated plant proteins encoded by a small gene family in each species. They are thought to act as a receptor for the auxin indole-3-acetic acid (141, 142, 300) and thereby to mediate a wide range of physiological responses including a reduction in cytoplasmic pH in certain cells (93). Analysis of the gene structure reveals a four-intron/five-exon arrangement, with the central, third exon encoding the region which includes the peptide responsible for binding the carboxylic acid group of indole-3-acetic acid. This motif, known as box A (41) or D16 (300), is now thought to be equivalent to the conserved motif 1 in the cupin notation (69), a finding supported by observations on two similar proteins isolated from shoot apices of peach (Prunus persica L. cv. Akatsuki) (217). These latter proteins have been designated ABP 19 (gi‖1916807) and ABP 20 (gi‖1916809) on the basis of their ability to bind auxin, albeit at low affinity (217). Recent analysis of their sequences shows a greater level of similarity to the GLPs (the closest neighbor [E value 3e-78] is GLP3 [gi‖1755164] from A. thaliana) than to any of the functionally better characterized ABPs.

EpimerasesAnother group of cupin enzymes involved in the synthesis of bacterial and archaeal cell wall components are the epimerases, such as dTDP-4-dehydrorhamnose 3,5-epimerase (also known as dTDP-l-rhamnose synthase) (EC 5.1.3.13 ), which converts dTDP-4-keto-6-deoxy-d-glucose into dTDP-4-keto-6-deoxy-l-mannose. These enzymes are about 185 aa in length and contain the two-motif cupin signature usually separated by a distance of 28 residues; both motifs contain a single globally conserved histidine residue. They are encoded byrfbC (or equivalent), part of the rfb gene cluster (160, 189, 193, 205, 276). Most rfboperons start with an rfbABCD cluster, which is responsible for the synthesis of TDP-rhamnose (184); this cluster is followed by rfbIFGH in organisms that produce 3,6-dideoxyhexoses.

These epimerases are located in the periplasm, and it is relevant to the theme of this review to note that periplasmic proteins are, as a rule, folded into stable, protease-resistant conformations, consistent with the digestive nature of this compartment (70).

Many of these capsular polysaccharides have potential economic importance as aqueous rheological control agents for diverse industrial and food applications. Such compounds include xanthan gum (Xanthomonas campestris) (22), and the sphingans (e.g., gellan, welan, and rhamsan) produced by species ofSphingomonas (314). It has been proposed that the various sphingans be thought of as defensive in nature, similar to the protective capsules (224, 249, 277, 297) of many invasive pathogenic bacteria (e.g. alginate).

AraC-Type Transcription FactorsOf all the bacterial transcriptional regulators, possibly the best characterized are the members of the AraC/XylS family (88). This family, named after its first member, AraC (a regulator of the arabinose pathway in E. coli), contains more than 100 members, which can be subdivided into various classes on a functional basis. These functions are associated primarily with carbon metabolism, stress responses, and pathogenesis, with the former category including factors that control the degradation of arabinose (AraC), cellobiose (CelD/ChbR), melibiose (MelR), raffinose (RafR), rhamnose (RhaR), and xylose (XylR).

Sequence analysis shows most members of this family to be 250 to 300 residues in length, comprising a conserved C-terminal of about 100 aa which binds DNA, and a nonconserved N-terminal domain which binds the effector molecule (44). There is much more information available on the DNA binding component, although the specific details of the N-terminal section (particularly of the AraC protein) are more relevant to the present review. This regulator has been subject to detailed structural (269, 270) and molecular (250, 255) analysis over several years. In summary, the N-terminal section comprises an arabinose-binding, eight-stranded β-barrel, which is joined to the DNA-binding domain via a linker region; the barrel-shaped section is also responsible for the dimerization of the molecule, a factor which determines its 3D shape and therefore its ability to bend the associated DNA strand. Close analysis of the sequence (90) and structure (Dunwell, unpublished) of this barrel-shaped element reveals a previously undetected similarity to the conserved β-barrel core of the cupin proteins (Fig.2). Of this related subgroup of regulators involved in sugar degradation, that showing the closest sequence similarity to GLPs and other cupins is CelD (221). This protein was named on the basis of its presumed involvement in the utilization of cellobiose, although recent studies (150) have shown that the real function is as a regulator in the catabolism of the disaccharide chitobiose; on that basis, its gene has been renamed chbR, part of the chb(N,N-diacetylchitobiose) operon. The significance of this reassignment is that it further supports a functional link both to the other bacterial enzymes concerned with sugar metabolism (e.g., PMIs and epimerases) and to the higher-plant cupins, particularly the sucrose-binding proteins (detailed below). In this context, there is an additional circumstantial link between chitobiose and cupins, in that vicilins from cowpea (Vigna unguiculata) are known to bind chitin (248), and it has been suggested that the vicilin-induced inhibition of yeast cell growth is due to binding of the protein to the chitin component of the cell walls (96, 97).

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Fig. 2.

Comparative structures of two orientations of the arabinose-binding domain of the AraC protein (above) and the two-domain phaseolin storage protein (below), showing the similar β-barrel element in the center of each domain, with associated α-helixes. The apparent gap in the E/F loop in phaseolin is due to the lack of resolution of the 3D structure at that point (177).

Gentisate 1,2-Dioxygenase and 1-Hydroxy-2-Naphthoate DioxygenaseIdentification of the two-domain composition of gentisate 1,2-dioxygenase (GDO) and 1-hydroxy-2-naphthoate dioxygenase (HNDO) is a novel finding made during the preparation of this review. The two enzymes are involved in the degradation of a range of related aromatic compounds, with the former enzyme, GDO (EC 1.13.11.4 ), catalyzing the oxygenolytic cleavage (between carbons 1 and 2) of gentisate (2,5-dihydroxybenzoate) to form maleylpyruvate, a compound that can be converted to central metabolites of the Krebs cycle either by cleavage to pyruvate and maleate or by isomerization to fumarylpyruvate and subsequent cleavage to fumarate and pyruvate. GDOs have been purified and characterized in many gram-positive and gram-negative bacteria (Klebsiella pneumoniae [143, 281],Moraxella osloensis [50],Sphingomonas [305], and Actinomycetales[109]), with possibly the best characterized such genes being those from species of Pseudomonas(110). For example, a GDO encoded by nagI(gi‖3406827) has recently been identified in P. aeruginosastrain U2 (86) and a very similar polypeptide (E value 3e-45) is encoded by nucleotides 5549669 to 5548674 of a contig (gnl‖PAGP_287‖Paeruginosa_Contig54) fromPseudomonas strain PAO1. Another very similar sequence (gi‖3293534) (Fig. 1) has also recently been found inHaloferax sp. strain D1227, an extreme halophile isolated from soil contaminated with highly saline oil brine and the only known aerobic archaeon able to utilize aromatic compounds as its sole carbon sources (85).

The only previous comment on the sequence similarity of these two types of dioxygenase was that made by Werwath et al. (305), who cloned the GDO gene gtdA (gi‖3550667) fromSphingomonas sp. strain RW5 and showed that its product had a low similarity to the HNDO (EC 1.13.11.38 ) (gi‖3288681) encoded by the phdI gene of the phenathrene-degradingNocardioides sp. strain Kp7 (134). This latter enzyme catalyzes the cleavage of 1-hydroxy-2-naphthoate totrans-2′-caboxybenzalpyruvate, a ring cleavage between the carboxylated and hydroxylated carbons analogous to that effected by GDO.

Both classes of enzyme described in this section have a multimeric structure; GDO has an apparent subunit molecular mass of 38 to 39 kDa and is claimed to have either a tetrameric (85, 281, 305) or hexameric (151) composition, whereas HNDO has a molecular mass of 45 kDa and is considered to be hexameric (134). Like most other dioxygenases of the extradiol class (those that cleave an aromatic ring adjacent to two vicinal hydroxyl groups), both GDO and HNDO contain 1 mol of Fe²⁺ per mol of subunit (those from Arthrobacter globiformis andBacillus brevis contain manganese, although they utilize the same coordinating residues). These features, namely, a tetrameric or hexameric composition and the presence of a transition metal in the active site, are shared with other cupin proteins described in this review, such as barley OXO, which is now known to contain manganese (238, 239; S. Bornemann, personal communication).

Oxalate DecarboxylasesAmong the many oxalate-degrading enzymes isolated from fungi, possibly the best characterized is that from the wood-rotting fungus Collybia velutipes. This particular homo-bicupin enzyme (intermotif spacing of 20 aa in each domain) degrades oxalate to formate and carbon dioxide and appears not to have any requirement for cofactors. It was therefore selected for use in strategies to reduce the levels of endogenous oxalate in plants (198, 199). The enzyme itself has an acidic pI, is stable over a wide pH range, is moderately thermostable, and has a molecular mass of 560 kDa as estimated by gel filtration and a subunit mass of 64 kDa before and 55 kDa after treatment with endo-β-N-acetylglucosaminidase, thus suggesting a glycosylated status (198). The sequence of the C. velutipes enzyme has been published as gi‖1604990 (52), and recently the sequence of a similar enzyme fromAspergillus phoenices was reported (C. J. Scelonge and D. L. Bidney, 1 October 1998, PCT patent application WO 98/42827). Presumed homologues of these sequences have also been identified (Dunwell, unpublished) (see below) in the bacterial species B. subtilis and Streptococcus mutans (encoded by nucleotides 555 to 1676 from contig 1009) (Fig.3).

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Fig. 3.

Alignment of the six 20+20 bicupin proteins (presumed OXDCs) from Streptococcus mutans (S. mut),Bacillus subtilis (B. sub1, YvrK; B. sub2, YoaN), Collybia velutipes (C. vel),Aspergillus phoenices (A. pho), andSynechocystis (Syn.) (see the text for details), showing the positions of the two conserved motifs (boxed) within each of the two domains. Residues conserved in all sequences are indicated with asterisks below the alignment; residues also conserved between the two domains are indicated with asterisks above the alignment. TheA. phoenices sequence (Scelonge and Bidney, PCT patent application WO 98/42827) has been amended by insertion of an additional nucleotide at residue 344 to correct a presumed frameshift error introduced during the sequencing of this gene.

Sucrose-Binding ProteinsAmong the two-domain relatives of the seed storage proteins is a sucrose-binding protein (SBP) (gi‖548900 and gi‖2765097) found at low abundance in the plasma membrane of cotyledons, leaves, and mature phloem of legumes (103); a similar sequence (gi‖2148163) from the cycad Zamia furfuracea is known (40). Recent comparison (219) of the soybean SBP sequence with that of vicilin has shown that the N-terminal domain of SBP contains 12 of the 13 residues conserved across the whole vicilin family, with the C-terminal domain having 10 of the 12 conserved residues.

Although the overall tertiary structure of SBP can be predicted by comparison to phaseolin, it is also possible that analysis of the disaccharide-binding domain of CelD/ChbR (see “AraC-type transcription factors” above) would provide further information on the specific ligands in the binding site.

Seed Storage ProteinsDuring the development of plant seeds there is a massive accumulation of nitrogen and carbon reserves in the form of proteins that can withstand desiccation and be used as a source of energy for the germinating embryo. In legumes, the globulin type of storage proteins can be divided into two forms, the legumins and the vicilins. The former are usually found as hexameric complexes (sedimentation coefficient, 11S), with each subunit derived from a precursor complex consisting of two domains, an N-terminal acidic α chain and a C-terminal basic β chain, which remain associated following proteolytic processing. The latter proteins occur as 7S trimers, with each subunit being a 50- to 70-kDa polypeptide that is subject to variable levels of processing. Examination of Fig. 1 shows that most of the storage proteins either lack any of the conserved His residues or contain a single conserved His in motif 1. It is presumed that, as a consequence, they have no metal-binding ligands and therefore no enzymatic activity. There is, however, a massive accumulation of oxalate (maximum 24% [dry weight]) during early seed development in soybean (131) and presumably in other legumes, and it is tempting to speculate on the possibility that this compound acts as a substrate for a residual oxalate-degrading capacity provided by the storage proteins being produced at that period. Knowledge of the tertiary structure of the two storage proteins phaseolin (177) and canavalin (155) and the finding of certain globally conserved residues (20) provided the basis for the generation of a homology model of wheat germin (90) and all subsequent predictions of cupin structures (Fig. 2).

In addition to the well-known major storage proteins found in seeds and spores (261), other, less abundant proteins of this type have been the subject of detailed analysis. Among the best characterized is the major peanut allergen Ara h1, a member of the vicilin family (43, 54, 258) and the protein responsible for the majority of cases of fatal food-induced anaphylaxis. In a recent study (258), it has been shown using molecular modelling that the 23 linear immunoglobulin E-binding epitopes cluster into two main regions, thus providing a rational target for transgenic approaches (66, 67) to modify the allergenic residues. Like many other members of the cupin family described in this review, the Ara h1 protein has a very high level of stability; it survives intact in most food-processing methods and also resists digestion by the gastrointestinal tract or its in vitro equivalent (23). It has been suggested (258) that this stability may be due to its compact structure, which limits the possibility for protease digestion and also facilitates its passage across the small intestine. It is presumed that these biophysical characteristics are shared by the allergenic single-domain GLP recently identified in ground black pepper (180).

Bicupins of Unknown FunctionAs described above, there is now good evidence for a wide variety of bicupins from archaeal species (e.g., the GDO fromHaloferax [85]), many bacteria includingB. subtilis and Streptococcus pyogenes (the 15+15 bicupin encoded by contig 272) and several eukaryotes (e.g., seed storage proteins). With the exception of the two classes of dioxygenase and the OXDCs from Collybia velutipes and Aspergillus phoenices, no biochemical function has yet been assigned to the microbial bicupins. It would be of particular interest to investigate the activities of the four examples from B. subtilis, which now probably represents the best organism for the study of prokaryotic cupin diversity. Within the higher plants, there is also evidence for another previously unidentified class of bicupins (e.g., theArabidopsis thaliana hypothetical gene gi‖2244827).

Overall Conservation of Cupin Motifs in Proteins Encoded by the B. subtilis GenomeAnalysis of the genome of B. subtilis, using the methods described above, identified a total of 20 sequences that fulfil, at least in part, the characteristic two-motif cupin signature. The alignment of this conserved section is given in Fig.4, which also shows the range of intermotif spacing (15 to 54 aa) as well as the overall protein size (113 to 432 aa). It can be seen that the sequences fall into several subgroups on the basis of their detailed similarity, with the great majority having the characteristic signature of three histidines (two in the first motif and one in the second), along with conserved proline and glycine residues in the second motif.

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Fig. 4.

Alignment of the conserved two-motif signature in the cupin proteins from B. subtilis, showing the GenBank identifier, the gene name, site in the genome (in kilobases from origin; sequences likely to be included in the section of the chromosome trapped in the prespore during septation are denoted by asterisks) (311), coding strand, details of the two motifs, total size of the protein, and its calculated pI. The sequences are subdivided on the basis of similarity. In the four two-domain proteins (YxaG, YwfC, YoaN, and YvrK), the first and second domains are designated a and b, respectively.

Particular reference must be made to YdaE (gi‖2632720), which is most unusual in having an additional six residues between strands C and D within motif 1. It also has a comparatively long intermotif distance.

Closest Neighbors and Possible FunctionsOnly two of the cupins in B. subtilis have designated names (PMI [phosphomannose isomerase] and SpsK [spore capsule synthesis K protein]); most of the sequences are so-called y genes (166), i.e., genes of unknown function that make up 70% of the total gene complement. The closest neighbor for each protein sequence, as estimated by a BlastP analysis, is given in Table2. In terms of function, it can be seen that the sequences can be divided into various subgroups that include five AraC-type transcription factors, three PMIs, and a cysteine dioxygenase. However, an obvious problem inherent in this type of comparison based on the total sequence is that it takes no account of the occurrence of multidomain proteins. For example, analysis of sections of the SpsK protein suggests that it probably represents a bifunctional enzyme similar to one from Actinobacillus actinomycetemcomitans, with an N-terminal domain presumed to have dDTP–4-dehydrorhamnose reductase activity (cf gi‖2650312 fromArchaeglobus fulgidus) and a C-terminal domain (containing the cupin element) with dTDP–4-dehydrorhamnose 3,5-epimerase activity (c.f. gi‖2622921 from Methanobacterium thermoautotrophicum).

The unusual protein YdaE is most closely related to a previously unidentified protein from Morganella morganii.

Additional confirmation of the different functional subgroups can be obtained by examination of the pI values given in Fig. 4. This shows that all the transcription factors have values between 6.10 to 8.48 whereas the other proteins (with the exception of YjlB and YrkC) are more acidic, with values between 4.41 and 5.90.

Domain StructureThere are 16 single-domain and 4 two-domain (bicupin) proteins encoded by the B. subtilis genome (Fig. 4). Bicupins are referred to below on the basis of their intermotif spacing (e.g., 15+15, 20+20). Of the former group of one-domain sequences, particular note should be made of the two examples that have a spacing of 20 residues, namely, YkrZ and YrkC. The former is most similar to a recently described sequence from the hyperthermophilic bacteriumAquifex aeolicus (53), whereas the second sequence is closer to a sequence from Prunus persica.

Probably the most interesting of the latter group of bicupins are the two sequences YoaN and YvrK, which have a very high level of similarity (E value 1e-130) to a sequence from Streptococcus mutans(contig 1009) and to the oxalate decarboxylases encoded by gi‖1604990 from Collybia velutipes, a wood-rotting basidiomycete (198), and the related sequence from Aspergillus phoenices (Scelonge and Bidney, patent application). These fungal enzymes are related to the Synechocystis protein gi‖1652630 (69), the only other 20+20 microbial bicupin identified to date. Detailed inspection of the six-sequence alignment provided in Fig. 3 reveals two main features. First, there are 64 (c. 16% of the total) globally conserved residues, mostly clustered within the two cupin motifs, which have the composition GX₂RX₂HWHX_3/4EWX₅G, and GX₁₀HX₄. Of these 64 residues, only 11 (ca. 3%), including the 3 histidines (90), also show conservation between the first and second domains. Second, the fungal OXDCs are more similar to the sequences from B. subtilis andS. mutans than they are to the Synechocystisprotein.

Additional alignments of protein sequence (data not shown) suggest that the most likely single-domain progenitor of the two-domain 20+20 proteins is YkrZ and that this protein is slightly more similar to YvrK than to YoaN. The evolutionary time course of events is thus indicated to be (YkrZ) × 2 → YvrK → YoaN. Similarly, it is likely that YjlB (18 spacing) is the progenitor of its closest neighbor, the two-domain YxaG (15+15) sequence (Table 2), although this would imply that the increase in intermotif spacing from 15 to 18 residues in YjlB occurred after the duplication event. It is also noticeable from the alignments of single cupins with their putative bicupin derivatives that the single-domain sequences (e.g., YjlB) always show a higher degree of similarity to the C-terminal domain than to the N-terminal domain of the respective bicupin (e.g., YxaG).

If alignments are based on the DNA rather than the protein sequence, additional features can be observed (Fig.5). For example, the doublet of bicupin genes (yvrK and yoaN) are very similar to each other (65% identity; E value 2.2e-72), although each gene has a different pattern of insertions and deletions (indels). However, these differences in nucleotide sequence do not disrupt the conserved two-motif regions; where there are indels within these motifs, they are equivalent in the two genes and do not alter the globally conserved residues.

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Fig. 5.

Alignment of the two 20+20 bicupin genes yoaN(gi‖2634260, denoted 4260) and yvrK(gi‖2635821, denoted 5821) from B. subtilis, showing the positions of the two conserved motifs within each of the two domains. Similar nucleotides are shown as dots, and deletions are shown as dashes. The deletions marked with asterisks in motif 1 of the first domain (1-bp deletion) and motif 2 of the second domain (2-bp deletion) denote two examples of the compensatory system of deletions and insertions that maintain the same reading frame for the two genes throughout the majority of the sequence. The deletions which produce a respective difference in the presence of a Gly residue (Fig. 3) are marked with vertical arrowheads. The statistical analysis is as follows: score = 1,706 (256.0 bits), expect = 2.2e-72, P = 2.2 e-72, identities = 756/1,151 (65%).

In an earlier study (69) it was suggested that the two-domain OXDC proteins may represent direct progenitors of the two-domain storage proteins. Recent phylogenetic evidence (260) now shows that the two duplication events occurred independently.

Physical Location of Cupin Genes within the B. subtilis ChromosomeThe cupin sequences are arranged on both DNA strands, and although they are distributed throughout the chromosome (Fig. 4), there is a possible increase in the kilobase value as the complexity of the protein increases. It is also noticeable that the two members of the doublet (yvrK and yoaN) are on opposite strands and that all four of the two-domain sequences are located in the second half of the chromosome (i.e., above kb 2000).

Size of Cupin Gene Families in Prokaryotes and EukaryotesIn general terms, it can be seen that the number of cupin genes varies from 2–7 in the archaeal genomes sequenced to date and inAquifex aeolicus (often considered to be the most deeply branching bacterium) to 15–20 in B. subtilis andSynechocystis (65) to >40 in angiosperms and animals (Dunwell, unpublished). For example, in Arabidopsis thaliana there are about 20 GLPs, which themselves can be further subdivided on the basis of sequence into five to seven subclasses. Apart from the identification of OXO activity in some of the cereal germins and in a related protein in Pinus caribaea(212), there is no functional information about these particular eukaryotic proteins.

Interestingly, with the exception of the epimerases (e.g., gi‖1666505 from Leptospira interrogans), cupins appear to be absent from spirochetes, as estimated by the analysis of the relatively small genomes of Borrelia burgorferi (genome size, 1.44 Mb) (79), Treponema pallidum (1.14 Mb) (80), Rickettsia prowazekii (the closest extant relative of the ancestor to mitochondria, with a genome size of 1.10 Mb) (9), and Chlamydia trachomatis (1.05 Mb) (274); for reference, the genome size of B. subtilis is 4.20 Mb. Presumably, this specific absence of cupins is part of the reductive evolution that leads to the general absence of any genes for cellular biosynthetic functions, including most aspects of cell wall synthesis, in these intracellular parasites.

It should also be noted that eukaryotic cupins are not restricted to fungi, yeasts, and plants. The latest estimate of their abundance in animals (68) suggests a widespread occurrence, withCaenorhabditis elegans having a total of at least 15 sequences (all single domain), including some (e.g., gi‖3877049) which have a 20-aa spacing and are very similar to sequences fromA. thaliana (gi‖2739365), rice (gi‖3201969), andH. sapiens (gi‖2822126). In terms of function, some of these animal sequences, such as PMIs, cysteine dioxygenases, and epimerases, are related to microbial sequences described above, while others, such as the CENP-C centromeric proteins (282) and various zinc finger transcription factors, are specific to eukaryotes.

Do Cupin Families Arise from Gene Duplication or Genome Fusion?In the discussion above, it has been assumed that the gene duplication events to generate doublets and higher-order multiples all took place in a single bacterial genome. However, there is a possibility that the present genome of B. subtilis is the result of one or more fusions of ancestral genomes (either in whole or in part) and that the doublets of genes represent the consequence of this fusion process (130). Large-scale analyses of protein sequences have already led to the suggestion that the evolution of the archaea included at least one major merger between ancestral cells from the bacterial lineage and the lineage leading to the eukaryotic nucleocytoplasm (158, 159, 240, 242). In the same context, it has also been proposed (105) that the eukaryotic nuclear genome is a chimera that has received major contributions from both an archaeal species and a gram-negative species. Such a network of fusion events would clearly lead to a complicated structure for many prokaryotic genomes, with gene doublets resulting from either fusion or internal duplication.

Physical Location of Cupin Genes in the Bacterial GenomeAn important aspect of cupin evolution concerns the fact that cupin genes are distributed on both DNA strands of the B. subtilis chromosome. This suggests that at some stage during evolution, there was a duplication of genes from one strand to the other. If the specific example of the 20+20 bicupins is examined, for example, it can be seen that in Synechocystis there is one such gene on the left strand whereas in B. subtilis the two closely similar genes are transcribed by different strands (Fig. 4).

Analysis of the location of the cupin sequences within the B. subtilis genome (Fig. 4) shows a general progression, with the genes encoding the shorter proteins being located closer to the origin of replication than are the genes encoding the longer proteins. Similarly, all the two-domain sequences are located in the second half of the genome (from kb 2038 to 4106). There is little information on any factor(s) that might determine the higher-order location of genes in this species, although it has been suggested (166) that the “grey hole” located at kb ∼600 might be related to the temporary chromosome partition observed during the first stages of the sporulation, when a segment of about one-third of the chromosome enters the prespore and remains the sole part of the chromosome in the prespore for a significant transition period (311). In light of the general link between cupin proteins and sporulation/desiccation (20, 69), it is possible that there is a functional reason for the clustering of genes required during this stage of the life cycle.

There are two additional pieces of evidence for physical linkage between related cupin genes and between such genes and other functionally related genes. Most interestingly, it seems likely that the recently sequenced genome of A. aeolicus (53) represents the earliest known stage of bacterial cupin evolution, a suggestion supported by the fact that the familyAquificaceae is the most deeply branching family within the bacterial domain on the basis of phylogenetic analysis of 16S rRNA sequences. Specifically, A. aeolicus genome contains three cupin genes. Between aq_528 and aq_529 there is a cryptic cupin gene encoding a protein with a 15-aa spacing similar to gi‖2128971 fromMethanococcus jannaschii, and in addition there are two other closely linked cupin genes (aq_1975 and aq_1978). The first encodes a protein with a 19-aa spacing (gi‖2984230), and the second encodes a protein with a 15-aa spacing (gi‖2984227); both have close relatives in B. subtilis (Table 2). It seems most likely, therefore, that aq_1975 was the product of duplication of aq_1978 and that it then diverged in sequence by the addition of 12 bp (encoding 4 aa) to the intermotif region while retaining its close physical position in the chromosome. In contrast, their two paralogs in B. subtilis (yjlB and ykrZ) are located more than 200 kb apart, presumably as a consequence of recombination.

In this context, there is also an interesting relationship between theydbB gene and its adjacent sequences in the B. subtilis genome. A TBlastN analysis of this gene shows that its closest relative from plants is the wheat protein germin, so named because of its high level of expression in germinating embryos (172). Adjacent to ydbB is gsiB (for “glucose starvation-inducible protein B”) (gi‖2632740), a sequence that has as its closest neighbours (E values 2e-18, and 2e-15, respectively) the plant Em protein (gi‖1169515) from A. thaliana (91) and the Lea (late embryogenesis abundant) protein (gi‖547819) from barley (75, 243). It is possible, therefore, that the two adjacent bacterial sequences have a common developmental link that began with a role in stress response, was retained throughout evolution, and is now associated with embryo development in higher plants.

Comparison of Single-Domain and Two-Domain CupinsDetailed analysis of various pairwise alignments of the single-domain and two-domain cupins show a number of interesting features relating to the possible origin and evolution of these proteins. First, as described above, single-domain proteins are more similar to the C-terminal domain than to the N-terminal domain of their respective two-domain relatives. The reason for this disparity is unknown, but it is possible that there is less selection pressure to conserve the sequence of the N-terminal domain in a two-domain cupin. Another possible explanation for such variation is suggested by similar discoveries in the family of extradiol dioxygenases, where there are also single- and double-domain members (74). Among some of the latter enzymes, there is evidence that the two domains express different phylogenies, suggesting the possibility that these particular enzymes arose from recombination or even fusion of genes encoding different dioxygenases.

Cupins and the Comparative Structure of Microbial Cell WallsThe very close similarity between the two-domain 20+20 bicupin (gi‖1652630) from the gram-negative cyanobacteriumSynechocystis (65) and the related sequences fromB. subtilis and Streptococcus mutans (Fig. 3) supports the observation (105) that the cyanobacteria constitute one of the deepest-branching clades within the gram-negative species and have a close affinity to the gram-positive species. Indeed, analysis quoted by Gupta (105) and based on sequences from glutamate dehydrogenase, phosphoribosyl formyl glycinamidine synthase, and some 16S rRNAs suggests a clade comprising the gram-positive bacteria, the cyanobacteria, and the archaea. Additional circumstantial evidence for a close link between some gram-positive bacteria and some archaea comes from an analysis of the archaeal cupins. Whereas only two such sequences have been identified in Methanococcus jannaschii (42), seven have been found inMethanobacterium thermoautotrophicum (Dunwell, unpublished). It may be that the additional cupins in M. thermoautotrophicum are linked to its particular cell wall characteristics, since it was noted by Gupta (105) that a number of archaea, including Methanobacterium, exhibited a thick and homogeneous cell wall that shows positive staining in the Gram reaction. Perhaps the M. thermoautotrophicum sequences with greatest relevance in the context of wall complexity are the two hypothetical proteins gi‖2621649 (89 aa) and gi‖2621650 (89 aa) that contain sequences with similarity to the first and second motifs of the spsK (for “spore capsule synthesis K” [gi‖2636317]) protein from B. subtilis. Indeed, close analysis of these two adjacent archaeal ORFs suggests that they are more likely to be contiguous sections of a single coding region, particularly since addition of a single nucleotide (to correct a frameshift) produces a modified, longer ORF encoding a polypeptide most similar (E value 3e-19) to the C-terminal 151 aa of this B. subtilis protein, a probable dTDP-4-dehydrorhamnose 3,5-epimerase (cf gi‖2622921). Such enzymes are involved in the rhamnose biosynthetic pathway linked to the production of complex exopolysaccharides in cell walls.

In the latest discussion of prokaryotic classification, Gupta (106) has used differences in cell wall architecture to argue against the concept of considering the Archaea(307) to be a separate kingdom and in favour of linking the gram-positive bacteria and the archaea in a grouping of monoderm prokaryotes (surrounded by a single membrane) distinct from the diderm prokaryotes (i.e., all true gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). It is possible that the functional role of many cupins in cell wall synthesis will help in the resolution of this debate.

Microbiological Significance of Oxalic Acid and Oxalate-Degrading EnzymesOxalic acid has been implicated in a wide range of environmental effects including several biological and geochemical processes in soils (71, 101). For example, oxalate is the major organic anion in many forest soils throughout the world, and as such it has a large effect on the availability of phosphorus, aluminum, and calcium (154). This is because oxalate will chelate aluminum and iron, thereby making more phosphorus available to plant roots. Interestingly, aluminum has recently been found to stimulate the production and secretion of oxalate from roots of buckwheat (188, 323) in addition to inducing the synthesis of a defence related GLP in wheat. As well as this role in plant nutrition, oxalic acid is linked to general weathering of soil minerals and the subsequent precipitation of insoluble metal oxalates (87). This latter process is associated with the survival of fungi growing in the presence of potentially toxic metal compounds (e.g., copper-containing wood preservatives). It has also been exploited in the solubilization of heavy metals from bauxite, clay, sand, sewage sludge, and other metal bearing materials. In many of these applications,Aspergillus niger is favored as the best species for oxalic acid production (251, 252, 279), in contrast to the medical dangers of such production by this organism.

A related environmental role for oxalic acid (and oxalate-degrading enzymes) comes from its key importance in the carbon cycle and the release of CO₂ from rotting wood (98, 99), a process largely mediated by basidiomycetous white rot fungi. Such fungi, along with their brown rot equivalents (203), are known to produce oxalic acid (72, 257) and oxalate-degrading enzymes (73, 202) under some conditions. The biochemical basis for the role of oxalate include its capacity to chelate manganese and thus to stimulate the activity of Mn peroxidase, an extracellular, glycosylated enzyme involved in lignin degradation (16, 163, 292). This enzyme is also implicated in the commercial use of the fungus Bjerkandera in the biobleaching of delignified craft pulp during paper making (201, 208, 236). In all applications of this type, the level of oxalate, and thus the practical value of the particular technology, depends upon the level of the OXDC (or equivalent) enzyme, and therefore the discovery of the putative structure of OXDC, albeit indirectly by virtue of its relationship to OXO and seed storage proteins (69), provides the first opportunity to consider directed modification of the enzyme.

It is also relevant to emphasize the key role of manganese in the chemical reactions utilized in both lignin synthesis (Mn²⁺is the metal ion present in the active site of OXO, which provides H₂O₂ required for cell wall cross-linking) (239) and lignin degradation (see the comments above on lignin peroxidase). Oxalate and manganese together therefore control a large segment of the carbon cycle which involves both the fixation of carbon in woody biomass and its later release from that material.

Role of Oxalate in Plant PathogenesisAs described below in the context of transgenic plants, oxalic acid is a toxin associated with many plant pathogens, particularlySclerotinia sclerotiorum (185) and related species. It is secreted into the plant during the infection process, is implicated in the degradation of the plant cells, and is then often reused by the pathogen or precipitated in the form of calcium oxalate crystals (78, 315).

Medical Diagnosis and TreatmentAs described above, the OXO and OXDC enzymes have great commercial importance in assays for oxalate in blood and urine (116, 123). Such assays are vital in the control of hyperoxaluria, particularly in patients suffering from urolithiasis, i.e., the formation of stones (crystals of calcium oxalate) in the urinary tract (245); this complaint afflicts more than 10% of the U.S. population. Excess oxalate levels in humans can have many causes, including hereditary intolerance to the normal levels of oxalate in the diet (100); consumption of ascorbate (13), ethylene glycol, diethylene glycol, or xylitol; inhalation of the anaesthetic methoxyflurane; and infection with Aspergillus(known as aspergillosis) (169, 226). Such levels are also found in cystic fibrosis patients as a consequence of antibiotic treatment that eliminates Oxalobacter formigenes from the gut flora (262) and in premature babies fed with infant formula rather than breast milk (124). In addition to dietary or pharmacological treatments to reduce metabolic oxalate levels (120, 121), other suggested means of reducing oxalate intake include the use of immobilized or encapsulated (18, 19) oxalate-degrading enzymes in the digestive tract or in the peritoneal cavity. For example, OXO which had been immobilized by adsorption onto chitosan and cross-linking with glutaraldehyde was shown to have enhanced resistance to proteolytic digestion and heavy-metal inactivation and was considered suitable for oral administration (231). In a related study (230), rats implanted in their peritoneal cavity with dialysis membranes containing banana OXO were able to metabolize intraperitoneally injected [¹⁴C]oxalate, as well as its precursor, [¹⁴C]glyoxalate. The leading therapeutic compound in preclinical development is probably IxC2-62/47 (from Ixion Biotechnology Inc.), an orally administered product consisting of two recombinant enzymes of bacterial origin (M. J. Allison and H. Sidhu, 26 November 1998, PCT patent application WO 98/52586).

As an alternative to treatment of patients with these conditions, it is possible to reduce the oxalate content of food prior to its being eaten (176; R. Yoshida, T. Tanaka, and Y. Hotta, 5 March 1994, European patent application 0639329 A1). In one such method, it was shown (132) that the oxalate-degrading bacteriumEubacterium lentum WYH-1, isolated from human feces (133), could completely degrade 1 mg of oxalate per ml in artificial intestinal juice or the oxalate in an infusion of black tea—one of the major sources of oxalate in the human diet (321). Similarly, barley roots in a stirred-tank reactor have been used to reduce the level of oxalate in ginger juice (313). The use of microorganisms or isolated enzymes for this purpose is the subject of several patent applications (G. Kohlbecker, F. Heinz, and R. Schildbach, 8 April 1982, German patent application). Transgenic approaches to reducing oxalate levels in plants will be considered below.

In addition to this relatively common metabolic disorder, there is a more infrequent but much more serious genetic condition (primary hyperoxaluria), which leads to deposition of calcium oxalate throughout the body, often with fatal consequences. Treatment of this condition is also discussed below.

Human Gene TherapyAs mentioned in the previous section, primary hyperoxaluria (55) is a potentially fatal condition. It exists in two forms, type 1, which is due to a defect in the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase/serine pyruvate aminotransferase, and type 2, which involves glyoxalate reductase/d-glycerate dehydrogenase, a cytosolic enzyme found in hepatocytes and leukocytes. At present, treatments include pharmacological approaches (121) or preemptive liver transplantation (149, 254). Aside from the implantation of immobilized OXO or the oral administration of recombinant enzymes, there has been a longstanding interest in the potential of gene therapy for treatment of these life-threatening conditions. Although their eventual aim was to clone and utilize the fungal oxalate decarboxylase gene (263; B. Finlayson and A. Peck, 3 November 1988, PCT patent application), success was first achieved in the cloning of the bacterial equivalent (186, 187), the oxalyl coenzyme A decarboxylase from Oxalobacter formigenes, an anaerobic oxalate-degrading bacterium found in the mammalian intestine (4, 15, 165). It is hoped that the increased understanding of the oxalate-degrading enzymes described in this review will soon lead to further advances in this important area of human therapy.

Bioremediation and Industrial UsesOxalate, particularly sodium oxalate, is a significant environmental contaminant in industrial processes such as the smelting of alumina by the Bayer process. At present, the disposal methods comprise either burning or burial in landfill sites. It is hoped that these methods may be replaced in future by use of various means of bacterial degradation involving either an alkalophilicBacillus species (209) or Pseudomonas oxalaticus (11), an organism isolated from rhubarb patches (a rich source of oxalate). It is possible that the recently described new species of obligately oxalotrophic Ammonophilus oxalaticus and A. oxalivorans (319) isolated from the rhizosphere of sorrel (Rumex acetosa, another species with a high oxalate content) could be used for these purposes.

A recent application in this area is the claimed use (N. O. Nilvebrandt, A. Reiman, and F. De Sousa, 26 February 1998, PCT patent application) of oxalate-degrading enzymes (OXO and/or OXDC) to reduce the levels of oxalate in the process liquids during the production of pulp and paper. Wood contains oxalic acid at concentrations of 0.1 to 0.4 kg/ton (bark contains up to 15 kg/ton), and during processing this compound can easily precipitate in the form of calcium oxalate crystals, which cause problems in pipework, washing filters, and heat exchangers. A similar previous application (117) involved the use of these enzymes to inhibit the deposition of beer stones during the brewing process. Levels of oxalate also must be kept low to prevent the problem of “gushing” (see also reference107) during this process.

Another potential industrial use of OXDC and related enzymes concerns the use of fungal exopolysaccharides. One such compound is scleroglucan, a neutral glucan produced by Sclerotinia glucanicum and composed of a linear chain of β-d(1,3)-linked d-glucopyranosyl residues with single d-glycopyranosyl residues linked β(1,6) to every third residue of the main chain. Because of its structure and high molecular weight, it has great value as a viscosifier in enhanced oil recovery, a role it which its closest rival is xanthan gum (122). Unfortunately, production of scleroglucan in reactors is accompanied by the concurrent synthesis of oxalate (302), an unwanted by-product that makes purification more costly. Such synthesis is stimulated at a pH above 3.5, partly because of inhibition of OXDC. It would therefore be most valuable to be able to modify the characteristics of this enzyme in order to reduce the level of oxalate formed during the production process.