Structural insights into substrate specificity of crotonase from the n-butanol producing bacterium Clostridium acetobutylicum

Crotonase from Clostridium acetobutylicum (CaCRT) is an enzyme that catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway. To investigate the molecular mechanism underlying n-butanol biosynthesis, we determined the crystal structures of the CaCRT protein in apo- and acetoacetyl-CoA bound forms. Similar to other canonical crotonase enzymes, CaCRT forms a hexamer by the dimerization of two trimers. A crystal structure of CaCRT in complex with acetoacetyl-CoA revealed that Ser69 and Ala24 to be signature residues of CaCRT, which results in a distinct ADP binding mode wherein the ADP moiety is bound at a different position compared with other crotonases. We also revealed that the substrate specificity of crotonase enzymes is determined by both the structural feature of the α3 helix region and the residues contributing the enoyl-CoA binding pocket. A tight formed α3 helix and two phenylalanine residues, Phe143 and Phe233, aid CaCRT to accommodate crotonyl-CoA as the substrate. The key residues involved in substrate binding, enzyme catalysis and substrate specificity were confirmed by site-directed mutagenesis.     

Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.     

Crystal structures of Paenibacillus polymyxa beta-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases

Bacteria species involved in degradation of cellulosic substrates produce a variety of enzymes for processing related compounds along the hydrolytic pathway. Paenibacillus polymyxa encodes two homologous beta-glucosidases, BglA and BglB, presenting different quaternary structures and substrate specificities. We previously reported the 3D-structure of BglA, which is highly specific against cellobiose. Here, we present structural analysis of BglB, a monomeric enzyme that acts as an exo-beta-glucosidase hydrolyzing cellobiose and cellodextrins of higher degree of polymerization. The crystal structure of BglB shows that several polar residues narrow the active site pocket and contour additional subsites. The structure of the BglB-cellotetraose complex confirms these subsites, revealing the substrate-binding mode, and shows the oligosaccharide-enzyme recognition pattern in detail. Comparison between BglA and BglB crystal structures suggests that oligomerization in BglA can assist in fine-tuning the specificity of the active centre by modulating the loops surrounding the cavity. We have solved the crystal structure of BglB with bound thiocellobiose, a competitive inhibitor, which together with the BglB-cellotetraose complex delineate the general features of the aglycon site. The detailed characterization of the atomic interactions at the aglycon site show a recognition pattern common to all bacterial beta-glucosidases, and presents some differences with the aglycon site in plant beta-glycosidases essentially by means of a different orientation of the basal Trp. The crystal structures of of BglB with a covalently bound inhibitor (derived from 2-fluoroglucoside) and glucose (produced by hydrolysis of the substrate in the crystal), provide additional pictures of the binding events and the intermediates formed during the reaction. Altogether, this information can assist in the understanding of subtle differences of the enzyme mechanism and substrate recognition within this family of enzymes, and consequently it can help in the development of new enzymes with improved activity or specificity.     

Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose

The beta-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-beta-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42). As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-beta-Gal at 1.6A and 2.2A resolution, respectively. A4-beta-Gal forms a homotrimeric structure resembling a flowerpot. Each monomer has an active site located inside a large central tunnel. The N-terminal domain of A4-beta-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis. The putative catalytic residues of A4-beta-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli beta-galactosidase. The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E.coli beta-galactosidase, but the recognition mechanism for a substrate is different. Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity. Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-beta-Gal structure. On the basis of the results of 1H NMR spectroscopy, A4-beta-Gal was determined to be a "retaining" enzyme. Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, beta-amylase.     

Mutational and structural analysis of aglycone specificity in maize and sorghum beta-glucosidases

Plant beta-glucosidases display varying substrate specificities. The maize beta-glucosidase isozyme Glu1 (ZmGlu1) hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOA-Glc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one), whereas the sorghum beta-glucosidase isozyme Dhr1 (SbDhr1) hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucoside). Structural data from cocrystals of enzyme-substrate and enzyme-aglycone complexes have shown that five amino acid residues (Phe198, Phe205, Trp378, Phe466, and Ala467) are located in the aglycone-binding site of ZmGlu1 and form the basis of aglycone recognition and binding, hence substrate specificity. To study the mechanism of substrate specificity further, mutant beta-glucosidases were generated by replacing Phe198, Phe205, Asp261, Met263, Phe377, Phe466, Ala467, and Phe473 of Glu1 by Dhr1 counterparts. The effects of mutations on enzyme activity and substrate specificity were studied using both natural and artificial substrates. The simple mutant replacing Phe198 by a valine had the most drastic effect on activity, because the capacity of this enzyme to hydrolyze beta-glucosides was almost completely abolished. The analysis of this mutation was completed by a structural study of the double mutant ZmGlu1-E191D,F198V in complex with the natural substrate. The structure reveals that the single mutation F198V causes a cascade of conformational changes, which are unpredictable by standard molecular modeling techniques. Some other mutations led to drastic effects: replacing Asp261 by an asparagine decreases the catalytic efficiency of this simple mutant by 75% although replacing Tyr473 by a phenylalanine increase its efficiency by 300% and also provides a new substrate specificity by hydrolyzing dhurrin.     

Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17

Crystal structures of chitosanase from Bacillus sp. K17 (ChoK) have been determined at 1.5 A resolution in the active form and at 2.0 A resolution in the inactive form. This enzyme belongs to the family GH-8, out of 93 glycoside hydrolase families, and exhibits the substrate specificity of subclass II chitosanase. The catalytic site is constructed on the scaffold of a double-alpha(6)/alpha(6)-barrel, which is formed by six repeating helix-loop-helix motifs. This structure is quite different from those of the GH-46 chitosanases and of GH-5. Structural comparison with CelA (a cellulase belonging to the same family GH-8) suggests that the proton donor Glu122 is conserved, but the proton acceptor is the inserted Glu309 residue, and that the corresponding Asp278 residue in CelA is inactivated in ChoK. The four acidic residues, Asp179, Glu309, Asp183 and Glu107, can be involved in substrate recognition through interactions with the amino groups of the glucosamine residues bound in the -3, -2, -1 and +1 sites, respectively. The hydrophobic Trp235, Trp166, Phe413 and Tyr318 residues are highly conserved for binding of the hexose rings at the -3, -2, +1 and +2 sites, respectively. These structural features indicate that enzymes in GH-8 can be further divided into three subfamilies. Different types of chitosanases are discussed in terms of convergent evolution from different structural ancestors.     

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.     

Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase

Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

Evolution of enzymatic activities in the enolase superfamily: structure of a substrate-liganded complex of the L-Ala-D/L-Glu epimerase from Bacillus subtilis

The members of the mechanistically diverse enolase superfamily share a bidomain structure formed from a (beta/alpha)7beta-barrel domain [a modified (beta/alpha)8- or TIM-barrel] and a capping domain formed from N- and C-terminal segments of the polypeptide. The active sites are located at the interface between the C-terminal ends of the beta-strands in the barrel domain and two flexible loops in the capping domain. Within this structure, the acid/base chemistry responsible for formation and stabilization of an enediolate intermediate derived from a carboxylate anion substrate and the processing of it to product is "hard-wired" by functional groups at the C-terminal ends of the beta-strands in the barrel domain; the identity of the substrate is determined in part by the identities of residues located at the end of the eighth beta-strand in the barrel domain and two mobile loops in the capping domain. On the basis of the identities of the acid/base functional groups at the ends of the beta-strands, the currently available structure-function relationships derived from functionally characterized members are often sufficient for "deciphering" the identity of the chemical reaction catalyzed by sequence-divergent members discovered in genome projects. However, insufficient structural information for liganded complexes for specifying the identity of the substrate is available. In this paper, the structure of the complex of L-Ala-L-Glu with the L-Ala-D/L-Glu epimerase from Bacillus subtilis is reported. As expected for the 1,1-proton transfer reaction catalyzed by this enzyme, the alpha-carbon of the substrate is located between Lys 162 and Lys 268 at the ends of the second and sixth beta-strands in the barrel domain. The alpha-ammonium group of the l-Ala moiety is hydrogen bonded to both Asp 321 and Asp 323 at the end of the eighth beta-strand, revealing a novel strategy for substrate recognition in the superfamily. The delta-carboxylate group of the Glu moiety is hydrogen bonded to Arg 24 in one of the flexible loops in the capping domain, thereby providing a structural explanation for the restricted substrate specificity of this epimerase [Schmidt, D. M., Hubbard, B. K., and Gerlt, J. A. (2001) Biochemistry 40, 15707-15715]. These studies provide important new information about the structural bases for substrate specificity in the enolase superfamily.     

Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.     

The central cavity from the (alpha/alpha)6 barrel structure of Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase contains two key histidine residues for reversible conversion

N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) catalyzes the reversible epimerization between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc). We report here the 2.0 A resolution crystal structure of the GlcNAc 2-epimerase from Anabaena sp. CH1. The structure demonstrates an (alpha/alpha)(6) barrel fold, which shows structural homology with porcine GlcNAc 2-epimerase as well as a number of glycoside hydrolase enzymes and other sugar-metabolizing enzymes. One side of the barrel structure consists of short loops involved in dimer interactions. The other side of the barrel structure is comprised of long loops containing six short beta-sheets, which enclose a putative central active-site pocket. Site-directed mutagenesis of conserved residues near the N-terminal region of the inner alpha helices shows that R57, H239, E308, and H372 are strictly required for activity. E242 and R375 are also essential in catalysis. Based on the structure and kinetic analysis, H239 and H372 may serve as the key active site acid/base catalysts. These results suggest that the (alpha/alpha)(6) barrel represents a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner alpha helices, thus forming a fine-tuned catalytic site in GlcNAc 2-epimerase.     

The 1.8 Å Resolution Structure of Hevamine,a Plant Chitinase/Lysozyme,and Analysis of the Conserved Sequence and Structure Motifs of Glycosyl Hydrolase Family 18

The three-dimensional structure of hevamine, a plant enzyme with chitinase and lysozyme activity, has been refined at 1.8 Å resolution to an R-factor of 14.9% and a freeR-factor of 19.6%. The final model consists of all 273 amino acid residues and 206 ordered water molecules. Two non-prolinecis-peptides were identified, involving Phe32 and Trp255, both of which are implicated in substrate binding.Other glycosyl hydrolase family 18 proteins with known three-dimen sional structure are bacterial chitinase A, endo-β-N-acetylglucosaminidase F₁, endo-β-N-acetylglucosaminidase H, and the two plant proteins concanavalin B and narbonin, which have no known enzymatic activity. All these structures contain a (βα)₈barrel fold, with the two family 18 consensus regions roughly corresponding to the third and fourth barrel strands. This confirms the grouping of these proteins into family 18, which was only based on weak and local sequence similarity. The substrate specificity of the enzymes is determined by the loops following the barrel strands that form the substrate binding site. All enzymes have an aspartic acid and a glutamic acid residue in positions identical with Asp 125 and the catalytic Glu127 of hevamine. The lack of chitinase activity of concanavalin B and narbonin can be explained by the absence of one of these carboxylate groups, and by differences in the loops that form the substrate-binding cleft in hevamine.     

Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

The crystal structure of the nitrile hydratase (NHase) from Bacillus smithii SC-J05-1 was determined. Our analysis of the structure shows that some residues that seem to be responsible for substrate recognition are different from those of other NHases. In particular, the Phe52 in the beta subunit of NHase from B. smithii covers the metal center partially like a small lid and narrows the active site cleft. It is well known that the NHase from B. smithii especially prefers aliphatic nitriles for its substrate rather than aromatic ones, and we can now infer that the Phe52 residue may play a key role in the substrate specificity for this enzyme. This finding leads us to suggest that substitution of these residues may alter the substrate specificity of the enzyme.     

X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.     

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Crystal structure and biochemical characterization of PhaA from Ralstonia eutropha, a polyhydroxyalkanoate-producing bacterium

PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the polyhydroxyalbutyrate (PHB) biosynthetic pathway and catalyzes the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA. To investigate the molecular mechanism underlying PHB biosynthesis, we determined the crystal structures of the RePhaA protein in apo- and CoA-bound forms. The RePhaA structure adopts the type II biosynthetic thiolase fold forming a tetramer by means of dimerization of two dimers. The crystal structure of RePhaA in complex with CoA revealed that the enzyme contained a unique Phe219 residue, resulting that the ADP moiety binds in somewhat different position compared with that bound in other thiolase enzymes. Our study provides structural insight into the substrate specificity of RePhaA. Results indicate the presence of a small pocket near the Cys88 covalent catalytic residue leading to the possibility of the enzyme to accommodate acetyl-CoA as a sole substrate instead of larger acyl-CoA molecules such as propionyl-CoA. Furthermore, the roles of key residues involved in substrate binding and enzyme catalysis were confirmed by site-directed mutagenesis.     

Structural basis for the substrate specificity of a Bacillus 1,3-1,4-beta-glucanase

Depolymerization of polysaccharides is catalyzed by highly specific enzymes that promote hydrolysis of the scissile glycosidic bond by an activated water molecule. 1,3-1,4-beta-Glucanases selectively cleave beta-1,4 glycosidic bonds in 3-O-substituted glucopyranosyl units within polysaccharides with mixed linkage. The reaction follows a double-displacement mechanism by which the configuration of the anomeric C(1)-atom of the glucosyl unit in subsite -I is retained. Here we report the high-resolution crystal structure of the hybrid 1,3-1,4-beta-glucanase H(A16-M)(E105Q/E109Q) in complex with a beta-glucan tetrasaccharide. The structure shows four beta-d-glucosyl moieties bound to the substrate-binding cleft covering subsites -IV to -I, thus corresponding to the reaction product. The ten active-site residues Asn26, Glu63, Arg65, Phe92, Tyr94, Glu105, Asp107, Glu109, Asn182 and Trp184 form a network of hydrogen bonds and hydrophobic stacking interactions with the substrate. These residues were previously identified by mutational analysis as significant for stabilization of the enzyme-carbohydrate complex, with Glu105 and Glu109 being the catalytic residues. Compared to the Michaelis complex model, the tetrasaccharide moiety is slightly shifted toward that part of the cleft binding the non-reducing end of the substrate, but shows previously unanticipated strong stacking interactions with Phe92 in subsite -I. A number of specific hydrogen-bond contacts between the enzyme and the equatorial O(2), O(3) and O(6) hydroxyl groups of the glucosyl residues in subsites -I, -II and -III are the structural basis for the observed substrate specificity of 1,3-1,4-beta-glucanases. Kinetic analysis of enzyme variants with the all beta-1,3 linked polysaccharide laminarin identified key residues mediating substrate specificity in good agreement with the structural data. The comparison with structures of the apo-enzyme H(A16-M) and a covalent enzyme-inhibitor (E.I) complex, together with kinetic and mutagenesis data, yields new insights into the structural requirements for substrate binding and catalysis. A detailed view of enzyme-carbohydrate interactions is presented and mechanistic implications are discussed.