Selective Immobilization of His-Tagged Phosphomannose Isomerase on Ni Chelated Nanoparticles with Good Reusability and Activity.

Self-stable precipitation polymerization was used to prepare an enzyme-immobilized microsphere composite. Phosphomannose isomerase (PMI) with His-tag was successfully immobilized on Ni2+ charged pyridine-derived particles. The maximum amount of PMI immobilized on such particles was ∼184 mg/g. Compared with free enzyme, the activity of the immobilized enzymes was significantly improved. In addition, the immobilized enzymes showed a much better thermostability than free enzymes. At the same time, the immobilized enzymes can be reused for multiple reaction cycles. We observed that the enzyme activity did not decrease significantly after six cycles. We conclude that the pyridine-derived particles can be used to selectively immobilize His-tagged enzymes, which can couple the enzyme purification and catalysis steps and improve the efficiency of enzyme-catalyzed industrial processes.

Encapsulation of Mannose-6-phosphate Isomerase in Yeast Spores and Its Application in l-Ribose Production.

A mannose-6-phosphate isomerase (MPI) from Geobacillus thermodenitrificans was expressed and successfully encapsulated into the Saccharomyces cerevisiae spores. Our results demonstrated that compared to the free enzyme, the MPI triple mutant encapsulated in osw2Δ spores exhibited much preferred enzymatic properties, such as enhanced catalytic activity, excellent reusability, thermostability, and tolerance to various harsh conditions. In combination with an l-arabinose isomerase (AI) also from G. thermodenitrificans, this technique of spore encapsulation was applied for producing a high-value rare sugar l-ribose from biomass-derived l-arabinose. Using a 10 mL reaction system, 350 mg of l-ribose was produced from 1 g of l-arabinose with a conversion yield of 35% by repeatedly reacting with 200 mg of AI-encapsulated spores and 300 mg of MPI-encapsulated spores. This study provides a very useful and concise approach for the synthesis of rare sugars and other useful compounds.

Footprints of natural selection at the mannose-6-phosphate isomerase locus in barnacles.

The mannose-6-phosphate isomerase (Mpi) locus in Semibalanus balanoides has been studied as a candidate gene for balancing selection for more than two decades. Previous work has shown that Mpi allozyme genotypes (fast and slow) have different frequencies across Atlantic intertidal zones due to selection on postsettlement survival (i.e., allele zonation). We present the complete gene sequence of the Mpi locus and quantify nucleotide polymorphism in S. balanoides, as well as divergence to its sister taxon Semibalanus cariosus We show that the slow allozyme contains a derived charge-altering amino acid polymorphism, and both allozyme classes correspond to two haplogroups with multiple internal haplotypes. The locus shows several footprints of balancing selection around the fast/slow site: an enrichment of positive Tajima's D for nonsynonymous mutations, an excess of polymorphism, and a spike in the levels of silent polymorphism relative to silent divergence, as well as a site frequency spectrum enriched for midfrequency mutations. We observe other departures from neutrality across the locus in both coding and noncoding regions. These include a nonsynonymous trans-species polymorphism and a recent mutation under selection within the fast haplogroup. The latter suggests ongoing allelic replacement of functionally relevant amino acid variants. Moreover, predicted models of Mpi protein structure provide insight into the functional significance of the putatively selected amino acid polymorphisms. While footprints of selection are widespread across the range of S. balanoides, our data show that intertidal zonation patterns are variable across both spatial and temporal scales. These data provide further evidence for heterogeneous selection on Mpi.

Structural and functional insights into phosphomannose isomerase: the role of zinc and catalytic residues.

Phosphomannose isomerase (PMI) is a housekeeping enzyme that is found in organisms ranging from bacteria to fungi to mammals and is important for cell-wall synthesis, viability and signalling. PMI is a zinc-dependent enzyme that catalyses the reversible isomerization between mannose 6-phosphate (M6P) and fructose 6-phosphate (F6P), presumably via the formation of a cis-enediol intermediate. The reaction is hypothesized to involve ring opening of M6P, the transfer of a proton from the C2 atom to the C1 atom and between the O1 and O2 atoms of the substrate, followed by ring closure resulting in the product F6P. Several attempts have been made to decipher the role of zinc ions and various residues in the catalytic function of PMI. However, there is no consensus on the catalytic base and the mechanism of the reaction catalyzed by the enzyme. In the present study, based on the structure of PMI from Salmonella typhimurium, site-directed mutagenesis targeting residues close to the bound metal ion and activity studies on the mutants, zinc ions were shown to be crucial for substrate binding. These studies also suggest Lys86 as the most probable catalytic base abstracting the proton in the isomerization reaction. Plausible roles for the highly conserved residues Lys132 and Arg274 could also be discerned based on comparison of the crystal structures of wild-type and mutant PMIs. PMIs from prokaryotes possess a low sequence identity to the human enzyme, ranging between 30% and 40%. Since PMI is important for the virulence of many pathogenic organisms, the identification of catalytically important residues will facilitate its use as a potential antimicrobial drug target.

Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-d-arabinonhydrazide.

Type I phosphomannose isomerases (PMIs) are zinc-dependent monofunctional metalloenzymes catalysing the reversible isomerization of d-mannose 6-phosphate to d-fructose 6-phosphate. 5-Phospho-d-arabinonhydrazide (5PAHz), designed as an analogue of the enediolate high-energy intermediate, strongly inhibits PMI from Candida albicans (CaPMI). In this study, we report the 3D crystal structure of CaPMI complexed with 5PAHz at 1.85 Å resolution. The high-resolution structure suggests that Glu294 is the catalytic base that transfers a proton between the C1 and C2 carbon atoms of the substrate. Bidentate coordination of the inhibitor explains the stereochemistry of the isomerase activity, as well as the absence of both anomerase and C2-epimerase activities for Type I PMIs. A detailed mechanism of the reversible isomerization is proposed.

Functional Replacement of Histidine in Proteins To Generate Noncanonical Amino Acid Dependent Organisms.

Simple strategies to produce organisms whose growth is strictly dependent on the presence of a noncanonical amino acid are useful for the generation of live vaccines and the biological containment of recombinant organisms. To this end, we report an approach based on genetically replacing key histidine (His) residues in essential proteins with functional His analogs. We demonstrate that 3-methyl-l-histidine (MeH) functionally substitutes for a key metal binding ligand, H264, in the zinc-containing metalloenzyme mannose-6-phosphate isomerase (ManA). An evolved variant, Opt5, harboring both N262S and H264MeH substitutions exhibited comparable activities to wild type ManA. An engineered Escherichia coli strain containing the ManA variant Opt5 was strictly dependent on MeH for growth with an extremely low reversion rate. This straightforward strategy should be applicable to other metallo- or nonmetalloproteins that contain essential His residues.

Simple and efficient synthesis of 5'-aryl-5'-deoxyguanosine analogs by azide-alkyne click reaction and their antileishmanial activities.

A series of non-hydrolysable 5'-aryl substituted GDP analogs has been synthesized by reacting 5'-azido-5'-deoxyguanosine with different aryl- and benzyloxy-alkynes. Cu(I) nanoparticles in water were found to be the most efficient catalyst, producing the desired 5'-arylguanosines with good yields. The synthesized compounds were screened for in vitro antileishmanial activity against Leishmania donovani axenic amastigotes and intramacrophage amastigotes stages. The 4-(3-nitrobenzyl)-1,2,3-triazole 5'-substituted guanosine analog was found to be the most active in the series with an IC50 of 8.6 µM on axenic amastigotes. Despite a rather low in vitro antileishmanial activity on the intramacrophage amastigotes, the absence of cytotoxicity on RAW 264.7 macrophages justifies further pharmacomodulations making this antileishmanial series promising.

Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi.

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD(+)-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP(+) as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.

Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities.

Characterization of a Mannose-6-Phosphate Isomerase from Bacillus amyloliquefaciens and Its Application in Fructose-6-Phosphate Production.

The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications.

BcPMI2, isolated from non-heading Chinese cabbage encoding phosphomannose isomerase, improves stress tolerance in transgenic tobacco.

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the L-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T1 seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H2O2 treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.

Protein similarity networks reveal relationships among sequence, structure, and function within the Cupin superfamily.

The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional clustering. Networks based only on cupin domains and networks based on the whole proteins provide complementary information. Domain-clustering supports phylogenetic conclusions that the N- and C-terminal domains of bicupin proteins evolved independently. Interestingly, although many functionally similar enzymatic cupin members bind the same active site metal ion, the structure and sequence clustering does not correlate with the identity of the bound metal. It is anticipated that the application of PSNs to this superfamily will inform experimental work and influence the functional annotation of databases.

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus.

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn(2+) that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K(m) and dramatic decrease of k(cat), and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.

Polarizable water networks in ligand-metalloprotein recognition. Impact on the relative complexation energies of Zn-dependent phosphomannose isomerase with D-mannose 6-phosphate surrogates.

Using polarizable molecular mechanics, a recent study [de Courcy et al. J. Am. Chem. Soc., 2010, 132, 3312] has compared the relative energy balances of five competing inhibitors of the FAK kinase. It showed that the inclusion of structural water molecules was indispensable for an ordering consistent with the experimental one. This approach is now extended to compare the binding affinities of four active site ligands to the Type I Zn-metalloenzyme phosphomannose isomerase (PMI) from Candida albicans. The first three ones are the PMI substrate ß-D-mannopyranose 6-phosphate (ß-M6P) and two isomers, α-D-mannopyranose 6-phosphate (α-M6P) and ß-D-glucopyranose 6-phosphate (ß-G6P). They have a dianionic 6-phosphate substituent and differ by the relative configuration of the two carbon atoms C1 and C2 of the pyranose ring. The fourth ligand, namely 6-deoxy-6-dicarboxymethyl-ß-D-mannopyranose (ß-6DCM), is a substrate analogue that has the ß-M6P phosphate replaced by the nonhydrolyzable phosphate surrogate malonate. In the energy-minimized structures of all four complexes, one of the ligand hydroxyl groups binds Zn(II) through a water molecule, and the dianionic moiety binds simultaneously to Arg304 and Lys310 at the entrance of the cavity. Comparative energy-balances were performed in which solvation of the complexes and desolvation of PMI and of the ligands are computed using the Langlet-Claverie continuum reaction field procedure. They resulted into a more favorable balance in favor of ß-M6P than α-M6P and ß-G6P, consistent with the experimental results that show ß-M6P to act as a PMI substrate, while α-M6P and ß-G6P are inactive or at best weak inhibitors. However, these energy balances indicated the malonate ligand ß-6DCM to have a much lesser favorable relative complexation energy than the substrate ß-M6P, while it has an experimental 10-fold higher affinity than it on Type I PMI from Saccharomyces cerevisiae. The energy calculations were validated by comparison with parallel ab initio quantum chemistry on model binding sites extracted from the energy-minimized PMI-inhibitor complexes. We sought to improve the models upon including explicit water molecules solvating the dianionic moieties in their ionic bonds with the Arg304 and Lys310 side-chains. Energy-minimization resulted in the formation of three networks of structured waters. The first water of each network binds to one of the three accessible anionic oxygens. The networks extend to PMI residues (Asp17, Glu48, Asp300) remote from the ligand binding site. The final comparative energy balances also took into account ligand desolvation in a box of 64 waters. They now resulted into a large preference in favor of ß-6DCM over ß-M6P. The means to further augment the present model upon including entropy effects and sampling were discussed. Nevertheless a clear-cut conclusion emerging from this as well as our previous study on FAK kinase is that both polarization and charge-transfer contributions are critical elements of the energy balances.

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant.

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.

The reaction mechanism of type I phosphomannose isomerases: new information from inhibition and polarizable molecular mechanics studies.

Type I phosphomannose isomerases (PMIs) are zinc-dependent metalloenzymes involved in the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). 5-Phospho-D-arabinonohydroxamic acid (5PAH), an inhibitor endowed with nanomolar affinity for yeast (Type I) and Pseudomonas aeruginosa (Type II) PMIs (Roux et al., Biochemistry 2004; 43:2926-2934), strongly inhibits human (Type I) PMI (for which we report an improved expression and purification procedure), as well as Escherichia coli (Type I) PMI. Its K(i) value of 41 nM for human PMI is the lowest value ever reported for an inhibitor of PMI. 5-Phospho-D-arabinonhydrazide, a neutral analogue of the reaction intermediate 1,2-cis-enediol, is about 15 times less efficient at inhibiting both enzymes, in accord with the anionic nature of the postulated high-energy reaction intermediate. Using the polarizable molecular mechanics, sum of interactions between fragments ab initio computed (SIBFA) procedure, computed structures of the complexes between Candida albicans (Type I) PMI and the cyclic substrate ß-D-mannopyranose 6-phosphate (ß-M6P) and between the enzyme and the high-energy intermediate analogue inhibitor 5PAH are reported. Their analysis allows us to identify clearly the nature of each individual active site amino acid and to formulate a hypothesis for the overall mechanism of the reaction catalyzed by Type I PMIs, that is, the ring-opening and isomerization steps, respectively. Following enzyme-catalyzed ring-opening of ß-M6P by zinc-coordinated water and Gln111 ligands, Lys136 is identified as the probable catalytic base involved in proton transfer between the two carbon atoms C1 and C2 of the substrate D-mannose 6-phosphate.

Synthesis and evaluation of non-hydrolyzable D-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-D-mannose as a new strong inhibitor of phosphomannose isomerases.

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs) from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-d-mannose and 6-deoxy-6-carboxymethyl-D-mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxymethyl-D-mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E. coli PMIs with K(m)/K(i) ratios of 28 and 8, respectively. We thus report the first malonate-based inhibitor of an aldose-ketose isomerase to date. Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy intermediate postulated for the isomerization reaction catalyzed by PMIs were also synthesized but behave as poor inhibitors of PMIs. A polarizable molecular mechanics (SIBFA) study was performed on the complexes of d-mannose 6-phosphate and two of its analogues with PMI from Candida albicans, an enzyme involved in yeast infection homologous to S. cerevisiae and E. coli PMIs. It shows that effective binding to the catalytic site occurs with retention of the Zn(II)-bound water molecule. Thus the binding of the hydroxyl group on C1 of the ligand to Zn(II) should be water-mediated. The kinetic study reported here also suggests the dianionic character of the phosphate surrogate as a likely essential parameter for strong binding of the inhibitor to the enzyme active site.

Cloning and characterization of phosphomannose isomerase from Sphingomonas chungbukensis DJ77.

Phosphomannose isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate in the extracellular polysaccharide (EPS) synthesis pathway. The gene encoding PMI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli. The pmi gene is 1,410 nucleotides long and the deduced amino acid sequence shares high homology with other bifunctional proteins that possess both PMI and GDP-mannose pyrophosphorylase (GMP) activities. The sequence analysis of PMI revealed two domains with three conserved motifs: a GMP domain at the N-terminus and a PMI domain at the C-terminus. Enzyme assays using the PMI protein confirmed its bifunctional activity. Both activities required divalent metal ions such as Co(2+), Ca(2+), Mg(2+), Ni(2+) or Zn(2+). Of these ions, Co(2+) was found to be the most effective activator of PMI. GDP-D-mannose was found to inhibit the PMI activity, suggesting feedback regulation of this pathway.

Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism.

Mannose-6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. X-ray crystal structures of MPI from Salmonella typhimurium in the apo form (with no metal bound) and in the holo form (with bound Zn2+) and two other structures with yttrium bound at an inhibitory site and complexed with Zn2+ and fructose 6-phosphate (F6P) were determined in order to gain insights into the structure and the isomerization mechanism. Isomerization involves acid/base catalysis with proton transfer between the C1 and C2 atoms of the substrate. His99, Lys132, His131 and Asp270 are close to the substrate and are likely to be the residues involved in proton transfer. The interactions observed at the active site suggest that the ring-opening step is probably catalyzed by His99 and Asp270. An active-site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. Zn2+ binding induces structural order in the loop consisting of residues 50-54. The metal atom appears to play a role in substrate binding and is probably also important for maintaining the architecture of the active site. Isomerization probably follows the previously suggested cis-enediol mechanism.

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides.

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the D- and L-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for D-lyxose isomerization was optimal at pH 7.0, 70 degrees C and 1 mM Co(2+). Under these conditions, the k(cat) and K(m) values were 74,300 s(-1) and 390 mM for D-lyxose and 28,800 s(-1) and 470 mM for L-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70 degrees C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of D-lyxose to D-xylulose with a 38% conversion yield after 3 h, and converted L-ribose to L-ribulose with a 29% conversion yield.