Refined structure of dienelactone hydrolase at 1.8A˚

Pathak, Dushyant; Ollis, David L.

doi:10.1016/0022-2836(90)90196-s

Cited by 128 publications

(80 citation statements)

References 60 publications

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“…Because of its close proximity to a presumedly conserved pentapeptide, Cys-60 was assumed to be the essential cysteine (17). Detailed X-ray crystallographic structure analyses of wild-type and mutant enzymes, however, proved this assignment to be wrong and showed that Cys-123 rather than Cys-60 is involved in the hydrolytic reaction (35,37). As in the case of the cysteine and serine proteases, the nucleophile is part of a catalytic triad, which consists of His-202 and Asp-171 in addition to Cys-123.…”

Section: Discussionmentioning

confidence: 99%

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Dienelactone hydrolase from Pseudomonas cepacia

et al. 1993

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(15,16,42,46,49). As a result of chloride elimination, the product, a cis-or trans-dienelactone (4-carboxymethylenebut-2-en-4-olide), carries an additional double bond as compared with muconolactone, the cycloisomerization product of unsubstituted cis,cis-muconate (Fig. 1). While muconolactone first must be converted to 3-oxoadipate enol-lactone to make lactone hydrolysis possible, the dienelactones already have an enol-lactone structure and can be hydrolyzed directly.Despite the overall structural similarity between 3-oxoadipate enol-lactone and the dienelactones, they are converted by different enzymes in Pseudomonas sp. strain B13 (46). The 3-oxoadipate enol-lactone hydrolase (EC 3.1.1.24) of this strain does not attack the dienelactones, whereas the dienelactone hydrolase converts both dienelactone isomers but not 3-oxoadipate enol-lactone. The B13 dienelactone hydrolase, which appears to be identical to the corresponding pAC27-encoded enzyme (17), was further characterized and found to resemble Pseudomonas putida 3-oxoadipate enol-lactone hydrolase with respect to molecular mass, inhibition by p-chloromercuribenzoate, amino acid composition, and NH2-terminal amino acid sequence (29). An identical pentapeptide, neighboring a cysteine residue in both hydrolases (17), was interpreted to represent a region conserved during divergent evolution of the hydrolases because of its proximity to an active-site thiol. However, detailed X-ray crystallographic studies of the pAC27-encoded dienelactone hydrolase suggested that Cys-123 rather * Corresponding author. than Cys-60, which neighbors the pentapeptide, is involved in lactone hydrolysis (35)(36)(37). This leaves the relationship between Pseudomonas sp. strain B13 dienelactone hydrolase and P. putida 3-oxoadipate enol-lactone hydrolase open to question.Recently, the investigation of enzymes induced during growth of several Alcaligenes strains and Pseudomonas cepacia with 4-fluorobenzoate demonstrated the existence of dienelactone hydrolases with quite different substrate specificities (45). Like the enzyme from Pseudomonas sp. strain B13, the pJP4-encoded dienelactone hydrolase of the 2,4-dichlorophenoxyacetate-utilizing strain Alcaligenes eutrophus JMP134 hydrolyzes both dienelactone isomers. These two enzymes, termed type III dienelactone hydrolases, are closely related to each other as indicated by 54.2% amino acid sequence identity between the pAC27-and pJP4-encoded hydrolases (39). The substrate specificity of another closely related protein, TcbE of Pseudomonas sp. strain P51 (50), is not yet known. In comparison, the dienelactone hydrolases (type I) of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes sp. strain A7 convert the trans-dienelactone much faster than the cis isomer (45). A dienelactone hydrolase (type II) with opposite specificity was induced by P. cepacia. We now report on the purification of the 3-oxoadipate enol-lactone hydrolase and the dienelactone hydrolase of P. cepacia and present some properties of the latter enzym...

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Section: Discussionmentioning

confidence: 99%

“…However, detailed X-ray crystallographic studies of the pAC27-encoded dienelactone hydrolase suggested that Cys-123 rather * Corresponding author. than Cys-60, which neighbors the pentapeptide, is involved in lactone hydrolysis (35)(36)(37). This leaves the relationship between Pseudomonas sp.…”

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confidence: 99%

Dienelactone hydrolase from Pseudomonas cepacia

et al. 1993

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“…The oxidation apparently occurred during X-ray exposure because mass spectrometric analysis of crystallized S112C before X-ray exposure indicated that Cys112 was not oxidized. Active site thiol oxidation by X-rays has been previously noted for cysteinyl hydrolases (44), including dienelactone hydrolase (45) and papain (46). Since dioxygen may be involved in the cysteine oxidation, we anaerobically crystallized S112C and collected diffraction data from the liquid nitrogen-frozen crystals maintained at 100K under a gaseous nitrogen stream.…”

Section: Crystal Structuresmentioning

confidence: 99%

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

et al. 2006

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Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD LB400 ) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 °C showed that the enzyme rapidly (1/τ 1 ∼ 500 s −1 ) transforms HOPDA (λ max = 434 nm) to a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/τ 2 = 54 s −1 , 1/τ 3 = 6 s −1 ∼ k cat ) with simultaneous biphasic appearance (48 and 8 s −1 ) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/τ 1 and 1/τ 2 , but affected neither 1/τ 3 nor k cat , suggesting that 1/τ 2 may reflect diffusive HPD dissociation, while 1/τ 3 represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and therefore postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (λ max = 492 nm). Crystal structures of wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 Å resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogenbonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.The microbial degradation of aromatic compounds is crucial to maintaining the global carbon cycle (1). Aerobic degradation typically involves oxygenation of the aromatic ring to produce a catechol followed by a dioxygenase-catalyzed ring-opening reaction. In one type of degradation pathway, ring-opening yields a meta-cleavage product (MCP) 1 . A serine hydrolase † This work was funded by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant) and the National Institutes of Health (GM-52381).*To whom correspondence should be addressed: Lindsay D. Eltis, leltis@interchange.ubc.ca, Phone: (604) Fax: (604)822 −6041. § These authors contributed equally to this work. ¶ Present addresses: JK: University of Kentucky, Lexington, KY 40506, USA; SD: Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, CO 80206; SS: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada 1 Abbreviations: MCP, meta-cleavage product; HPD, 2-hydroxy-penta-2,4-dienoic acid; PCB, polychlorinated biphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid; DHB, 2,3-dihydroxybiphenyl; DHBD, dihydroxybiphenyl dioxygenase; S k , ketonized substrate; E:S k , enzyme-bound ketonized substrate; P...

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“…Structures for several other enzymes of the ,B-ketoadipate pathway exist: P. aeruginosa protocatechuate 3,4-dioxygenase (a mixed/~-barrel enzyme) (Ohlendorf, Lipscomb & Weber, 1988), P. putida muconolactone isomerase (a unique decameric enzyme) (Katti, Katz & Wyckoff, 1989), and Pseudomonas sp. B13 dienelactone hydrolase (an ot/~ hydrolase fold structure) (Pathak & Ollis, 1990;Ollis et al, 1992).…”

Section: International Union Of Crystallographymentioning

confidence: 99%

Crystallization and preliminary crystallographic analysis of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa

Glumoff

Helin²,

Mazur³

et al. 1996

Acta Cryst D

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Crystals of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE; E.C. 5.5.1.5) from Neurospora crassa that diffract to high resolution have been obtained. The crystals belong to the orthorhombic space group P212121 with unit-cell dimensions a = 92.1, b = 159.7, c = 236.6A (at 103 K) and diffract at most to 2,~ resolution. The asymmetric unit of the crystals appears to contain two tetrameric CMLE molecules making up a total of 328 kDa per asymmetric unit. Both cross-linking with glutaraldehyde and cryo-cooling to 103 K have been used to facilitate data collection because the crystals are unstable in the X-ray beam; both techniques extend the crystal lifetime but cryo-cooling, unlike glutaraldehyde cross-linking, does not lower the quality of the diffraction pattern.

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Refined structure of dienelactone hydrolase at 1.8A˚

Cited by 128 publications

References 60 publications

Dienelactone hydrolase from Pseudomonas cepacia

Dienelactone hydrolase from Pseudomonas cepacia

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

Crystallization and preliminary crystallographic analysis of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa

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