The 1.3 A crystal structure of human mitochondrial Delta3-Delta2-enoyl-CoA isomerase shows a novel mode of binding for the fatty acyl group

The crystal structure of Delta3-Delta2-enoyl-CoA isomerase from human mitochondria (hmEci), complexed with the substrate analogue octanoyl-CoA, has been refined at 1.3 A resolution. This enzyme takes part in the beta-oxidation of unsaturated fatty acids by converting both cis-3 and trans-3-enoyl-CoA esters (with variable length of the acyl group) to trans-2-enoyl-CoA. hmEci belongs to the hydratase/isomerase (crotonase) superfamily. Most of the enzymes belonging to this superfamily are hexamers, but hmEci is shown to be a trimer. The mode of binding of the ligand, octanoyl-CoA, shows that the omega-end of the acyl group binds in a hydrophobic tunnel formed by residues of the loop preceding helix H4 as well as by side-chains of the kinked helix H9. From the structure of the complex it can be seen that Glu136 is the only catalytic residue. The importance of Glu136 for catalysis is confirmed by mutagenesis studies. A cavity analysis shows the presence of two large, adjacent empty hydrophobic cavities near the active site, which are shaped by side-chains of helices H1, H2, H3 and H4. The structure comparison of hmEci with structures of other superfamily members, in particular of rat mitochondrial hydratase (crotonase) and yeast peroxisomal enoyl-CoA isomerase, highlights the variable mode of binding of the fatty acid moiety in this superfamily.     

The crystal structure of delta(3)-delta(2)-enoyl-CoA isomerase

The active-site geometry of the first crystal structure of a Delta(3)-Delta(2)-enoyl-coenzyme A (CoA) isomerase (the peroxisomal enzyme from the yeast Saccharomyces cerevisiae) shows that only one catalytic base, Glu158, is involved in shuttling the proton from the C2 carbon atom of the substrate, Delta(3)-enoyl-CoA, to the C4 atom of the product, Delta(2)-enoyl-CoA. Site-directed mutagenesis has been performed to confirm that this glutamate residue is essential for catalysis. This Delta(3)-Delta(2)-enoyl-CoA isomerase is a hexameric enzyme, consisting of six identical subunits. It belongs to the hydratase/isomerase superfamily of enzymes which catalyze a wide range of CoA-dependent reactions. The members of the hydratase/ isomerase superfamily have only a low level of sequence identity. Comparison of the crystal structure of the Delta(3)-Delta(2)-enoyl-CoA isomerase with the other structures of this superfamily shows only one region of large structural variability, which is in the second turn of the spiral fold and which is involved in defining the shape of the binding pocket.     

Mutation of Lys242 allows Delta3-Delta2-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Delta3-Delta2-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k(cat) value of 1.0 s(-1) for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.     

Mutation of Lys242 allows Δ-Δ-enoyl-CoA isomerase to acquire enoyl-CoA hydratase activity

We report here a novel example of generating hydratase activity through site-directed mutagenesis of a single residue Lys242 of rat liver mitochondrial Δ³-Δ²-enoyl-CoA isomerase, which is one of the key enzymes involved in fatty acid oxidation and a member of the crotonase superfamily. Lys242 is at the C-terminal of the enzyme, which is far from the active site in the crotonase superfamily and forms a salt bridge with Asp149. A variety of mutant expression plasmids were constructed, and it was observed that mutation of Lys242 to nonbasic residues allowed the mutants to have enoyl-CoA hydratase activity confirmed by HPLC analysis of the incubation mixture. Kinetic studies of these mutants were carried out for both isomerase and hydratase activities. Mutant K242C showed a k_cat value of 1.0 s^− 1 for hydration reaction. This activity constitutes about 10% of the total enzyme activity, and the remaining 90% is its natural isomerase activity. To the best of our knowledge, this is the first report on the generation of functional promiscuity through single amino acid mutation far from the active site. This may be a simple and efficient approach to designing a new enzyme based on an existing template. It could perhaps become a general methodology for facilitating an enzyme to acquire a type enzymatic activity that belongs to another member of the same superfamily, by interrupting a key structural element in order to introduce ambiguity, using site-directed mutagenesis.     

The 4-oxalocrotonate tautomerase family of enzymes: how nature makes new enzymes using a beta-alpha-beta structural motif

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers. The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy. Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base. In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies. Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink. Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50. 4-OT is also the title enzyme of the 4-OT family of enzymes. The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif. The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position. Characterization of representative members has uncovered mechanistic and structural diversity. A new activity, a trans-3-chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities. Two new structures have also been found, along with the 4-OT hexamer. The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer. Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity. Amplification of this activity could have produced the full-fledged dehalogenase. The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes.     

A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2

2-Enoyl-CoA hydratase 2, a part from multifunctional enzyme type 2, hydrates trans-2-enoyl-CoA to 3-hydroxyacyl-CoA in the (3R)-hydroxy-dependent route of peroxisomal beta-oxidation of fatty acids. Unliganded and (3R)-hydroxydecanoyl coenzyme A-complexed crystal structures of 2-enoyl-CoA hydratase 2 from Candida tropicalis multifunctional enzyme type 2 were solved to 1.95- and 2.35-A resolution, respectively. 2-Enoyl-CoA hydratase 2 is a dimeric, alpha+beta protein with a novel quaternary structure. The overall structure of the two-domain subunit of eukaryotic 2-enoyl-CoA hydratase 2 resembles the homodimeric, hot dog fold structures of prokaryotic (R)-specific 2-enoyl-CoA hydratase and beta-hydroxydecanoyl thiol ester dehydrase. Importantly, though, the eukaryotic hydratase 2 has a complete hot dog fold only in its C-domain, whereas the N-domain lacks a long central alpha-helix, thus creating space for bulkier substrates in the binding pocket and explaining the observed difference in substrate preference between eukaryotic and prokaryotic enzymes. Although the N- and C-domains have an identity of <10% at the amino acid level, they share a 50% identity at the nucleotide level and fold similarly. We suggest that a subunit of 2-enoyl-CoA hydratase 2 has evolved via a gene duplication with the concomitant loss of one catalytic site. The hydrogen bonding network of the active site of 2-enoyl-CoA hydratase 2 resembles the active site geometry of mitochondrial (S)-specific 2-enoyl-CoA hydratase 1, although in a mirror image fashion. This arrangement allows the reaction to occur by similar mechanism, supported by mutagenesis and mechanistic studies, although via reciprocal stereochemistry.     

Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB

The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.     

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.     

Domain swapping in the low-similarity isomerase/hydratase superfamily: the crystal structure of rat mitochondrial Delta3, Delta2-enoyl-CoA isomerase

Two monofunctional Delta(3), Delta(2)-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the beta-oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.     

Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.     

Characterization of Cg10062 from Corynebacterium glutamicum: implications for the evolution of cis-3-chloroacrylic acid dehalogenase activity in the tautomerase superfamily

A 149-amino acid protein designated Cg10062 is encoded by a gene from Corynebacterium glutamicum. The physiological function of Cg10062 is unknown, and the gene encoding this protein has no obvious genomic context. Sequence analysis links Cg10062 to the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) family, one of the five known families of the tautomerase superfamily. The characterized tautomerase superfamily members have two distinctive characteristics: a beta-alpha-beta structure motif and a catalytic amino-terminal proline. Pro-1 is present in the Cg10062 amino acid sequence along with His-28, Arg-70, Arg-73, Tyr-103, and Glu-114, all of which have been implicated as critical residues for cis-CaaD activity. The gene for Cg10062 has been cloned and the protein overproduced, purified, and subjected to kinetic and mechanistic characterization. Like cis-CaaD, Cg10062 functions as a hydratase: it converts 2-oxo-3-pentynoate to acetopyruvate and processes 3-bromopropiolate to a species that inactivates the enzyme by acylation of Pro-1. Kinetic and (1)H NMR spectroscopic studies also show that Cg10062 processes both isomers of 3-chloroacrylic acid at low levels with a clear preference for the cis isomer. Pro-1 is critical for the dehalogenase and hydratase activities because the P1A mutant no longer catalyzes either reaction. The presence of the six key catalytic residues and the hydratase activity coupled with the absence of an efficient cis-CaaD activity and the lack of isomer specificity implicate factors beyond this core set of residues in cis-CaaD catalysis and specificity. This work sets the stage for in-depth mechanistic and structural studies of Cg10062, which could identify the additional features necessary for a fully active and highly specific cis-CaaD. Such results will also shed light on how cis-CaaD emerged in the tautomerase superfamily because Cg10062 could be characteristic of an intermediate along the evolutionary pathway for this dehalogenase.     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalyzed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalyzed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate dehalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalyzing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalyzed the conversion to 4-hydroxybenzoate with concomitant release of coenzyme A.     

A unique fungal lysine biosynthesis enzyme shares a common ancestor with tricarboxylic acid cycle and leucine biosynthetic enzymes found in diverse organisms

Fungi have evolved a unique α-aminoadipate pathway for lysine biosynthesis. The fungal-specific enzyme homoaconitate hydratase from this pathway is moderately similar to the aconitase-family proteins from a diverse array of taxonomic groups, which have varying modes of obtaining lysine. We have used the similarity of homoaconitate hydratase to isopropylmalate isomerase (serving in leucine biosynthesis), aconitase (from the tricarboxylic acid cycle), and iron-responsive element binding proteins (cytosolic aconitase) from fungi and other eukaryotes, eubacteria, and archaea to evaluate possible evolutionary scenarios for the origin of this pathway. Refined sequence alignments show that aconitase active site residues are highly conserved in each of the enzymes, and intervening sequence sites are quite dissimilar. This pattern suggests strong purifying selection has acted to preserve the aconitase active site residues for a common catalytic mechanism; numerous other substitutions occur due to adaptive evolution or simply lack of functional constraint. We hypothesize that the similarities are the remnants of an ancestral gene duplication, which may not have occurred within the fungal lineage. Maximum likelihood, neighbor joining, and maximum parsimony phylogenetic comparisons show that the α-aminoadipate pathway enzyme is an outgroup to all aconitase family proteins for which sequence is currently available. Received: 7 October 1997     

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.     

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

Determination of the chlorine kinetic isotope effect on the 4-chlorobenzoyl-CoA dehalogenase-catalyzed nucleophilic aromatic substitution

The chlorine kinetic isotope effect (KIE) on the dehalogenation of 4-chlorobenzoyl-CoA catalyzed by 4-chlorobenzoyl-CoA dehalogenase has been measured at room temperature and optimal pH. The measured value of (37)k = 1.0090 +/- 0.0006 is larger than the KIEs recently measured for haloalkane and fluoroacetate dehalogenase. This indicates that the transition state for dissociation of chloride ion from the Meisenheimer intermediate is sensitive to the chlorine isotopic substitution. Simple modeling suggests that this sensitivity originates in the high isotopic sensitivity of the C-Cl bond bending modes.     

Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.