Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.     

α,β‐Unsaturated Aldehydes as Substrates for Asymmetric C?C Bond Forming Reactions with Thiamin Diphosphate (ThDP)‐Dependent Enzymes

The enzymes benzaldehyde lyase (BAL) from Pseudomonas fluorescens, benzoylformate decarboxylase (BFD) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Saccharomyces cerevisiae provide different C C bond forming possibilities of α,β‐unsaturated aldehydes with aliphatic and aromatic aldehydes. Structure elucidation and determination of the absolute configuration of the products, which were obtained with high regio‐ and stereoselectivity were carried out. Selective 1,2‐reactivity with yields of 75% and >98% ee, for one single isomer ( A ) were obtained, by choosing the suitable enzyme in combination with the appropriate substrates. By varying enzymes or substrates the regioisomeric hydroxy ketones C , with up to >99% ee, can be obtained. The application of these new chiral building blocks in the synthesis of natural products or biological active substances is considerably facilitated by applying the different ThDP‐dependent enzymes as catalysts. Abbreviations: BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; PDC, pyruvate decarboxylase; His, hexahistidine; 2‐HPP, 2‐hydroxy‐1‐phenylpropan‐1‐one; PAC, phenylacetylcarbinol; NTA, nitrilotriacetic acid; ThDP, thiamin diphosphate; wt, wild‐type.     

Factors mediating activity, selectivity, and substrate specificity for the thiamin diphosphate-dependent enzymes benzaldehyde lyase and benzoylformate decarboxylase

Benzaldehyde lyase from Pseudomonas fluorescens and benzoylformate decarboxylase from Pseudomonas putida are homologous thiamin diphosphate-dependent enzymes that catalyze carboligase and carbolyase reactions. Both enzymes catalyze the formation of chiral 2-hydroxy ketones from aldehydes. However, the reverse reaction has only been observed with benzaldehyde lyase. Whereas benzaldehyde lyase is strictly R specific, the stereoselectivity of benzoylformate decarboxylase from P. putida is dependent on the structure and orientation of the substrate aldehydes. In this study, the binding sites of both enzymes were investigated by using molecular modelling studies to explain the experimentally observed differences in the activity, stereo- and enantioselectivity and substrate specificity of both enzymes. We designed a detailed illustration that describes the shape of the binding site of both enzymes and sufficiently explains the experimental effects observed with the wild-type enzymes and different variants. These findings demonstrate that steric reasons are predominantly responsible for the differences observed in the (R)-benzoin cleavage and in the formation of chiral 2-hydroxy ketones.     

Multiple modes of active center communication in thiamin diphosphate-dependent enzymes

Detection of interaction between cofactors at the active centers of homodimeric and homotetrameric enzymes is usually elusive by steady-state kinetic approaches and requires protein variants where such interactions are diminished or exaggerated. In this Account, evidence for active-center interactions will be presented for the following thiamin diphosphate-dependent enzymes: yeast pyruvate decarboxylase, benzoylformate decarboxylase, and examples from the 2-oxoacid dehydrogenase multienzyme complex class. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound. Perturbations that reveal active center communication include use of chromophoric substrates and substitutions of active center residues on putative pathways.     

An activity, stability and selectivity comparison of propioin synthesis by thiamine diphosphate-dependent enzymes in a solid/gas bioreactor

Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.     

Engineering the xenobiotic substrate specificity of maize glutathione S-transferase I

Glutathione S-transferases (GSTs) are a heterogeneous family of enzymes that catalyse the conjugation of glutathione (GSH) to electrophilic sites on a variety of hydrophobic substrates. In the present study three amino acid residues (Trp12, Phe35 and Ile118) of the xenobiotic binding site (H-site) of maize GST I were altered in order to evaluate their contribution to substrate binding and catalysis. These residues are not conserved and hence may affect substrate specificity and/or product dissociation. The results demonstrate that these residues are important structural moieties that modulate an enzyme's catalytic efficiency and specificity. Phe35 and Ile118 also participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. The effect of temperature on the catalytic activity of the wild-type and mutant enzymes was also investigated. Biphasic Arrhenius and Eyring plots for the wild-type enzyme showed an apparent transition temperature at 35 degrees C, which seems to be the result of a change in the rate-limiting step of the catalytic reaction. Thermodynamic analysis of the activity data showed that the activation energy increases at low temperatures, whereas the entropy change seems to be the main determinant that contributes to the rate-limiting step at high temperatures.     

Enantioselective C?C Bond Ligation Using Recombinant Escherichia coli‐Whole‐Cell Biocatalysts

Thiamine diphosphate (ThDP)‐dependent enzymes like benzaldehyde lyase from Pseudomonas fluorescens (BAL) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are versatile biocatalysts for the C C bond ligation of aldehydes to form enantiomerically pure 2‐hydroxy ketones. However, the large‐scale application of this enzyme class is often restricted by the required external addition of the expensive cofactor ThDP, as well as by the common use of dimethyl sulfoxide (DMSO) as a cosolvent, which leads to problems during the work‐up procedure. In the present paper we demonstrate that the addition of the excess cofactors, ThDP and magnesium ions (Mg²⁺), is not required when BAL or BFD are used in Escherichia coli resting cells. Furthermore, the combination of these resting cells with a biphasic reaction medium [methyl tert‐butyl ether (MTBE)/aqueous buffer] allows an increase of the substrate concentration up to 1 M, and an efficient extractive work‐up. As a practical example, e.g., the synthesis of (R)‐2‐hydroxy‐3,3‐dimethoxy‐phenylpropanone from benzaldehyde and 2,2‐dimethoxyacetaldehyde was optimized, achieving an isolated yield of 78 %, and an enantiomeric excess of 98 % ee in 24 h when operating at a substrate concentration of 0.4 M. The described reaction system in a biphasic medium is suitable for a wide range of aldehydes as substrates. The biphasic reaction medium minimizes also the formation of by‐products, which were observed when this reaction was performed in the conventional DMSO/buffer system.     

Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries

The directed evolution of proteins has benefited greatly from site-specific methods of diversification such as saturation mutagenesis. These techniques target diversity to a number of chosen positions that are usually non-contiguous in the protein's primary structure. However, the number of targeted positions can be large, thus leading to impractically large library size, wherein almost all library variants are inactive and the likelihood of selecting desirable properties is extremely small. We describe a versatile combinatorial method for the partial diversification of large sets of residues. Our library oligonucleotides comprise randomized codons that are flanked by wild-type sequences. Adding these oligonucleotides to an assembly PCR of wild-type gene fragments incorporates the randomized cassettes, at their target sites, into the reassembled gene. Varying the oligonucleotides concentration resulted in library variants that carry a different average number of mutated positions that comprise a random subset of the entire set of diversified codons. This method, dubbed Incorporating Synthetic Oligos via Gene Reassembly (ISOR), was used to create libraries of a cytosine-C5 methyltransferase wherein 45 individual positions were randomized. One library, containing an average of 5.6 mutated residues per gene, was selected, and mutants with wild-type-like activities isolated. We also created libraries of serum paraoxonase PON1 harboring insertions and deletions (indels) in various areas surrounding the active site. Screening these libraries yielded a range of mutants with altered substrate specificities and indicated that certain regions of this enzyme have a surprisingly high tolerance to indels.     

Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis

A structure-function analysis of the icosahedral RNA bacteriophagefr coat protein (CP) assembly was undertaken using linker-insertion,deletion and substitution mutagenesis. Mutations were specificallyintroduced into either pre-existing or artificially createdrestriction enzyme sites within fr CP gene expressed in Escherichiacoli from a recombinant plasmid. This directs synthesis of wildtype protein that undergoes self-assembly and forms capsid-likeparticles indistinguishable morphologically and immunologicallyfrom native phage particles. A series of fr CP variants containingsequence alterations in the regions which are (i) exposed onthe external surface of capsid or (ii) located on the contactingareas between CP subunits were obtained and their assembly propertiesinvestigated. The majority of mutants demonstrated reductionof assembly ability and formed either CP dimers (mutations atresidues 2, 10, 63 or 129) or both dimer and capsid structures(residue 2 or 69). The exceptions were variants demonstratingnormal assembly and containing insertions at residues 2, 50or 129 of thefr CP. A third type of assembled structure wasformed by a variant with a single amino acid substitution I104T.The aA-helix region (residues 97-111) is particularly sensitiveto mutation and any alteration in this region decreases accumulationof mutant protein in E.coli. The relative contributions of particularfr CP domains in maintenance of capsid structural integrityas well as the possible capsid assembly mechanism are discussed.     

Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)

Lecithin cholesterol acyltransferase (LCAT) is an interfacialenzyme active on both high-density (HDL) and low-density lipoproteins(LDL). Threading alignments of LCAT with lipases suggest thatresidues 50–74 form an interfacial recognition site andthis hypothesis was tested by site-directed mutagenesis. The(56–68) deletion mutant had no activity on any substrate.Substitution of W61 with F, Y, L or G suggested that an aromaticresidue is required for full enzymatic activity. The activityof the W61F and W61Y mutants was retained on HDL but decreasedon LDL, possibly owing to impaired accessibility to the LDLlipid substrate. The decreased activity of the single R52A andK53A mutants on HDL and LDL and the severer effect of the doublemutation suggested that these conserved residues contributeto the folding of the LCAT lid. The membrane-destabilizing propertiesof the LCAT 56–68 helical segment were demonstrated usingthe corresponding synthetic peptide. An M65N–N66M substitutiondecreased both the fusogenic properties of the peptide and theactivity of the mutant enzyme on all substrates. These resultssuggest that the putative interfacial recognition domain ofLCAT plays an important role in regulating the interaction ofthe enzyme with its organized lipoprotein substrates.     

Making tissue-type plasminogen activator more fibrin specific

The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296–299or DDD 364–366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466–470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe k_cat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296–299, where the presenceof fibrin manifests significant increases in both k_cat and K_m.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity.     

Chemical modification and site-directed mutagenesis of Tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8

3-Isopropylmalate dehydrogenase from an extreme thermo-phile,Thermus thermophUus HB8, was chemically modified with tetranitromethanewhich nitrated 1.5-2.0 Tyr residues per subunit. The nitrationwas biphask and parallel to the loss of activity. The modifiedresidue in the first phase was identified to be Tyr36, whichis distantly located from the active site of the enzyme. Thefunction of Tyr36 was investigated by site-specific replacementwith Phe. The Michaelis constant for the substrate or co-enzymewas not altered by the replacement, whereas the catalytic constantdecreased down to -5%. X-ray analysis of the mutant enzyme revealedthat Arg94 moved the largest distance among the active siteresidues, that is, the NH1 and NH2 of the guanidino group moved1.11 and 1.32 Å respectively. The results suggest thatArg94 is responsible for the enzyme catalysis     

Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin

The functional contributions of amino acid residues Thr218 andAsp304 of chymosin, both of which are highly conserved in theaspartic proteinases, are analysed by means of site-directedmutagenesis. The optimum pH values, milk-clotting (C) and proteolytic(P) activities and kinetic parameters for synthetic oligopeptidesas substrates were examined for the mutant enzymes. The mutationThr2l8Ser caused a marked increase in the C/P ratio, which seemedto be due to a change in substrate recognition. Although thenegative charge of Asp304 had been expected to play a role inlowering the optimum pH values in the aspartic proteinases,this turned out not to be the case in chymosin because boththe mutations Asp304Ala and Asp304Glu caused a similar shiftof the optimum pH towards the acidic side. In addition, themutation Lys220Leu, which we generated previously, was foundto cause a decrease in the C/P ratio, mainly due to the increasein the proteolytic activity.     

Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library

A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.     

Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum

The plasmepsins are involved in the degradation of host cell hemoglobin during malaria infection. Plasmepsin I (PM I) initiates the degradative process, and has been suggested as an attractive target for the treatment of malaria. The production of active recombinant PM I, however, has been challenging. We report for the first time, the expression and partial characterization of soluble recombinant PM I from Plasmodium falciparum in which a truncated form of PM I (Lys77P-Leu329) (P indicates a propart residues) was fused to thioredoxin in the pET32b(+) vector, Trx-tPM I and expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The soluble fusion protein was purified from cell culture using a combination of Ni(2+) affinity and gel filtration chromatography and was capable of autocatalytic activation at pH 4.0-5.5, which occurred at Leu116P-Ser117P, seven residues upstream of the native cleavage site (Gly123P-Asn1). The mature tPM I (mtPM I) was capable of hydrolyzing both human hemoglobin with a pH optimum of pH 2.8-4.0 and the synthetic fluorogenic peptide EDANS-CO-CH(2)-CH(2)-CO-ALERMFLSFP-Dap(DABCYL)-OH with a dual pH optima of pH 2.5-3.0 and pH 4.5-5.5. Using the synthetic substrate, mtPM I exhibited kinetic parameters comparable to native PM I.     

Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans

The role of four aromatic residues (W85, Y172, W266 and W274)in the structure–function relationship in xylanase A fromStreptomyces lividans (XlnA) was investigated by site-directedmutagenesis where each residue was subjected to three substitutions(W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four aminoacids are highly conserved among family 10 xylanases and structuraldata have implicated them in substrate binding at the activesite. Far-UV circular dichroism spectroscopy was used to showthat the overall structure of XlnA was not affected by any ofthese mutations. High-performance liquid chromatographic analysisof the hydrolysis products of birchwood xylan and xylopentaoseshowed that mutation of these aromatic residues did not alterthe enzyme's mode of action. As expected, though, it did reducethe affinity of XlnA for birchwood xylan. A comparison of thekinetic parameters of different mutants at the same positiondemonstrated the importance of the aromatic nature of W85, Y172and W274 in substrate binding. Replacement of these residuesby a phenylalanine resulted in mutant proteins with a K_M closerto that of the wild-type protein in comparison with the othermutations analyzed. The kinetic analysis of the mutant proteinsat position W266 indicated that this amino acid is importantfor both substrate binding and efficient catalysis by XlnA.These studies also demonstrated the crucial role of these activesite aromatic residues for the thermal stability of XlnA.     

Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel

CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.     

Modification of substrate-binding site of glutamyl endopeptidase from Bacillus intermedius

Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family. Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear. The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system). In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213-Thr) and studied its catalytic properties. Such modification proved not to affect the primary specificity of the enzyme. The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times). The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity.