Tamao Hisano

Crystal Structure of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL AN α/β HYDROLASE STRUCTURE THAT IS DIFFERENT FROM THE α/β HYDROLASE FOLD

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 Å resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an α/β structure formed by a six-stranded parallel β-sheet flanked by five α-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal β-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an α/β-type hydrolase, it does not belong to the α/β hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

Crystal structures of reaction intermediates of L-2-haloacid dehalogenase and implications for the reaction mechanism.

Crystal structures ofl-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, l-2-chlorobutyrate,l-2-chloro-3-methylbutyrate, orl-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-Å resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a d-configuration at the C2atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10–Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91–Asp-102 and Leu-117–Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme’s nucleophile Asp-10.

Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

Crystal Structure of the Oxygenase Component (HpaB) of the 4-Hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8

The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase is involved in the initial step of the 4HPA degradation pathway and catalyzes 4HPA hydroxylation to 3,4-dihydroxyphenylacetate. This enzyme consists of two components, an oxygenase (HpaB) and a reductase (HpaC). To understand the structural basis of the catalytic mechanism of HpaB, crystal structures of HpaB from Thermus thermophilus HB8 were determined in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4HPA. Structural analysis revealed that the binding and dissociation of flavin are accompanied by conformational changes of the loop between β5 and β6 and of the loop between β8 and β9, leading to preformation of part of the substrate-binding site (Ser-197 and Thr-198). The latter loop further changes its conformation upon binding of 4HPA and obstructs the active site from the bulk solvent. Arg-100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4a-hydroperoxyflavin intermediate.

Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity

The two‐component enzyme, 4‐hydroxyphenylacetate 3‐monooxygenase, catalyzes the conversion of 4‐hydroxyphenylacetate to 3,4‐dihydroxyphenylacetate. In the overall reaction, the oxygenase component (HpaB) introduces a hydroxyl group into the benzene ring of 4‐hydroxyphenylacetate using molecular oxygen and reduced flavin, while the reductase component (HpaC) provides free reduced flavins for HpaB. The crystal structures of HpaC from Thermus thermophilus HB8 in the ligand‐free form, the FAD‐containing form, and the ternary complex with FAD and NAD+ were determined. In the ligand‐free form, two large grooves are present at the dimer interface, and are occupied by water molecules. A structural analysis of HpaC containing FAD revealed that FAD has a low occupancy, indicating that it is not tightly bound to HpaC. This was further confirmed in flavin dissociation experiments, showing that FAD can be released from HpaC. The structure of the ternary complex revealed that FAD and NAD+ are bound in the groove in the extended and folded conformation, respectively. The nicotinamide ring of NAD+ is sandwiched between the adenine ring of NAD+ and the isoalloxazine ring of FAD. The distance between N5 of the isoalloxazine ring and C4 of the nicotinamide ring is about 3.3 Å, sufficient to permit hydride transfer. The structures of these three states are essentially identical, however, the side chains of several residues show small conformational changes, indicating an induced fit upon binding of NADH. Inactivity with respect to NADPH can be explained as instability of the binding of NADPH with the negatively charged 2′‐phosphate group buried inside the complex, as well as a possible repulsive effect by the dipole of helix α1. A comparison of the binding mode of FAD with that in PheA2 from Bacillus thermoglucosidasius A7, which contains FAD as a prosthetic group, reveals remarkable conformational differences in a less conserved loop region (Gly83–Gly94) involved in the binding of the AMP moiety of FAD. These data suggest that variations in the affinities for FAD in the reductases of the two‐component flavin‐diffusible monooxygenase family may be attributed to difference in the interaction between the AMP moiety of FAD and the less conserved loop region which possibly shows structural divergence. Proteins 2008.

CRYSTALLIZATION AND PRELIMINARY X-RAY CRYSTALLOGRAPHIC STUDIES OF L-2 HALOACID DEHALOGENASE FROM PSEUDOMONAS SP. YL

The dimeric L‐2‐haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n‐propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 Å, b = 62.78 Angst; c = 50.84 Å, and β = 122.4°, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 Å resolution.

A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions

Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys151, Asp306, and His335) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.

Crystallization and preliminary X-ray analysis of the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.

The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4 M 1,6-hexanediol, 0.1 M sodium acetate pH 5.0 and 25% (v/v) glycerol and diffracted X-rays to a resolution of 1.60 A. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 91.8, b = 99.6, c = 131.1 A. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V(M) of 2.8 A3 Da(-1) and a solvent content of 55.1%. Platinum-derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays to a resolution of 2.50 A. MAD data were successfully collected for structural determination using these crystals.

Crystallization and preliminary X-ray analysis of (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis.

Dimeric (R)-specific enoyl-coenzyme A (CoA) hydratase from Aeromonas caviae catalyzes the hydration of trans-2-enoyl-CoAs with carbon lengths of 4-6 to yield their corresponding (R)-3-hydroxyacyl-CoAs and is essential for polyhydroxyalkanoate (PHA) biosynthesis. The enzyme has been crystallized by vapour diffusion against a reservoir solution containing 20% polyethylene glycol 4000, 5% 2-propanol and 20 mM HEPES pH 7.0 at 298 K. Crystals belong to the monoclinic space group C2, with unit-cell parameters a = 111.54 (3), b = 59.29 (1), c = 47.27 (4) A, beta = 113.04 (2) degrees and contain a dimeric molecule in the asymmetric unit. Flash-cooling of a crystal at 100 K alters its unit-cell parameters to a = 109.82 (7), b = 57.98 (6), c = 46.84 (2) A, beta = 112.71 (3) degrees. Native data to a resolution of 1.7 A have been collected with 94.5% completeness and an R(merge) of 4.0% under cryogenic (100 K) conditions using synchrotron radiation.

Crystal structure of N-acylamino acid racemase from Thermus thermophilus HB8.

N-acylamino acid racemase (NAAAR) catalyzes the racemization of a broad range of N-acylamino acids and requires a divalent metal ion for expressing its activity.1,2 Because enantiopure amino acids are of industrial interest as chiral building blocks for antibiotics, herbicides, and drugs, the thermal stabilization of NAAAR has been undertaken by protein engineering based on its threedimensional structure to obtain an enzyme with more desirable industrial properties.3 NAAAR is a member of the enolase superfamily, which includes enzymes catalyzing a wide variety of reactions and performing diverse roles in the metabolism of living systems.4,5 All enzymes of the enolase superfamily utilize a common reaction mechanism, in which an enolate anion intermediate is generated by the abstraction of the a-proton from a carboxylate substrate by a catalytic residue and is stabilized by a divalent metal ion.6 The enolase superfamily is divided into subgroups based on sequence clustering and the identity of the catalytic residue.7 o-Succinylbenzoate synthase (OSBS) catalyzes the syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4cyclohexadiene-1-carboxylate to produce 4-(20-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. NAAAR and OSBS belong to the muconate lactonizing enzyme subgroup.7 In this group, two conserved lysine residues are located at the active site of these enzymes so that they can sandwich their substrates. In the NAAAR reaction, two lysine residues participate in a two-base-mediated reversible 1,1-proton exchange reaction.4,8 In the OSBS reaction, one of two conserved lysine residues functions as the only acid/base catalyst that abstracts the a-proton from the substrate and facilitates the departure of the bhydroxyl group in the syn-dehydration reaction.8 The other lysine residue likely provides electrostatic stabilization of the enediolate anion intermediate.8 Recently, it was demonstrated that NAAAR from Amycolatopsis sp. T1-60 exhibits not only the NAAAR activity but also the OSBS activity.9 Moreover, in addition to the NAAAR/ OSBS enzyme from Amycolatopsis, it has been suggested that other NAAARs from Deinococcus radiodurans and Thermus thermophilus might also be bifunctional enzymes, even though genes encoding proteins related to the menaquinone biosynthetic pathway are not found in these genomes.3,7 Here we report the crystal structure of NAAAR from T. thermophilus HB8.