Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway

Nandhagopal, N.; Yamada, Akira; Hatta, Takashi; Masai, Eiji; Fukuda, Masao; Mitsui, Yukio; Senda, Toshiya

doi:10.1006/jmbi.2001.4737

Cited by 54 publications

(67 citation statements)

References 43 publications

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“…The Nε2 atom of His265 forms a hydrogen bond with Oδ1 of Asn111 instead of the Oγ atom of Ser112 in subunit A and does not form any hydrogen bond in subunit B. Consistent with these data, the catalytic His and Ser are not hydrogen bonded in the BphD RHA1 and CarC J3 structures (17,19). The His and Ser are hydrogen bonded in the MhpC structure, but the bond is longer (3.3 Å) than typically observed in serine hydrolases (20).…”

Section: Crystal Structuressupporting

confidence: 62%

“…Considerably smaller surface areas of 1135 and 964 Å 2 were calculated for the AB' interface and the AA' interface, respectively. The A-B dimer interaction involves the β8 strands from both subunits forming an antiparallel β pair, and corresponds to the 1−5 interaction of the BphD RHA1 octamer (17). Similar interactions were also found in CumD IP01 (18), CarC J3 (19), and MhpC (20), suggesting the A-B dimer is a basic structural unit from which some members of this family, such as BphD LB400 and BphD RHA1 , may construct higher oligomers.…”

Section: Crystal Structuresmentioning

confidence: 65%

“…By contrast, unexplained electron density was observed adjacent to the Oγ atom of the active site serine in BphD RHA1 (17) and certain structures of MhpC (20), suggesting covalent modification. In the structure of the S112C variant of BphD LB400 crystallized aerobically, the electron density suggests that Cys112 is oxidized to the sulfonic acid in subunit A and to the sulfenic acid in subunit B (data not shown).…”

Section: Crystal Structuresmentioning

confidence: 82%

“…The active site can be divided into two sub-sites, P (polar) and NP (non-polar), located on each side of the catalytic serine. The P and NP sub-sites bind the dienoate moiety and phenyl ring of the substrate, respectively, and correspond to the proximal and distal parts identified in a study of BphD RHA1 (17). The P sub-site is lined with polar groups from the side chains of residues that are conserved in MCP hydrolases: Asn51, Asn111, Arg190, and Trp266.…”

Section: Crystal Structuresmentioning

confidence: 99%

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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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Section: Crystal Structuressupporting

confidence: 62%

Section: Crystal Structuresmentioning

confidence: 65%

Section: Crystal Structuresmentioning

confidence: 82%

Section: Crystal Structuresmentioning

confidence: 99%

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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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“…Pseudomonas putida TodF (23) and XylF (24), Pseudomonas azelaica HbpD (25), Rhodococcus sp. EtbDs (26) and BphD (27), Pseudomonas fluorescens CumD (28, 29), and E. coli MhpC (30). Their substrates are ketonic ring fission products of aromatic compounds and are hydrolytically converted to smaller molecules.…”

Section: Discussionmentioning

confidence: 99%

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

Fujii

Yasuoka

Tsai

et al. 2004

Journal of Biological Chemistry

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The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalenemelanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp 352 -His 380 -Ser 257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser 257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1 -carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser 257 -O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.

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Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

et al. 2005

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Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2',3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.

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Crystal structure of 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid (HPDA) hydrolase (BphD enzyme) from the Rhodococcus sp. strain RHA1 of the PCB degradation pathway

Cited by 54 publications

References 43 publications

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

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

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

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