Catalytic Mechanism of the Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Prokop, Zbyněk; Monincová, Marta; Chaloupková, Radka; Klvaňa, Martin; Nagata, Yasunobu; Janssen, Dick B.; Damborský, Jiřı́

doi:10.1074/jbc.m307056200

Cited by 88 publications

(86 citation statements)

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“…The resulting inhibitors are expected to bind into the active site in a way similar to that of the substrates, but they cannot undergo enzyme-catalyzed dehalogenation because they either contain strong carbon-fluorine bonds or are not amenable to nucleophilic displacement fore tested enzymes, but they exhibited significant inhibition effect only at millimolar concentrations. Such a finding is in a good agreement with mechanistic analysis of the HLD reaction mechanism, showing binding constants in the millimolar range (43). The most potent of the six tested molecules was 1-fluorohexane, which had an IC 50 of 5.4 mM toward DbjA.…”

Section: Discussionsupporting

confidence: 74%

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Buryška

Daniel

Kunka

et al. 2016

Appl Environ Microbiol

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bHaloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 M and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization. Haloalkane dehalogenases (HLDs; EC 3.5.1.8) are enzymes that catalyze the hydrolytic cleavage of carbon-halogen bonds in alkyl halides ( Fig. 1) with a wide range of potential applications in biocatalysis, biodegradation, biosensing, decontamination, and cell imaging (1). In structural terms, they belong to the ␣/␤-hydrolase superfamily (2-4). The HLDs have broad substrate specificity, enabling them to catalyze conversion of diverse chlorinated, brominated and iodinated alkanes, alkenes, cycloalkanes, alcohols, epoxides, carboxylic acids, esters, ethers, amides, and nitriles (5, 6).The first known members of this family were isolated from the bacteria Xanthobacter autotrophicus GJ10, Sphinghomonas paucimobilis UT26, and Rhodococcus rhodochrous NCIMB13064 (3, 7, 8), which colonize environments that have been heavily contaminated with halogenated pollutants, such as 1,2-dichloroethane, 1,2,3,4,5,6-hexachlorocyclohexane, and 1-chlorobutane. In these microorganisms, the HLDs were found to be components of metabolic pathways that enable the microbes to utilize otherwise toxic haloalkanes as their sole source of carbon and energy. The HLDs have been recently discovered in a wider range of organisms, including symbiotic bacteria such as Bradyrhizobium japonicum USDA110 and Mesorhizobium loti MAF303099 (9, 10), pathogenic bacteria such as Agrobacterium tumefaciens C58 and Mycobacterium spp. (11,12), and the eukaryotic organism Strongylocentrotus purpuratus (13). Even though HLDs have been studied intensively over the last 25 years and isolated from many different environments and species, most of their biological functions remain elusive. For instance, it is undoubtedly interesting that the plant pathogen Agrobacterium tumefaciens C58 carries the HLDencoding gene datA on its tumor-inducing plasmid while there is no clear link between HLD activity and tumorigenesis (1...

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Section: Discussionsupporting

confidence: 74%

Discovery of Novel Haloalkane Dehalogenase Inhibitors

Buryška

Daniel

Kunka

et al. 2016

Appl Environ Microbiol

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“…MI1205 and Sphingobium japonicum UT26, respectively, that can cleave the carbon-halogen bond in ␤-HCH. Haloalkane dehalogenases belong to the ␣/␤-hydrolase family, and their catalytic mechanism consists of the following steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond in the substrate and formation of an intermediate covalently bound to the nucleophile, (iii) hydrolysis of the alkyl-enzyme intermediate, and (iv) release of halide ion and alcohol (6). LinB MI and LinB UT share 98% sequence identity, with only 7 different amino acid residues (at positions 81, 112, 134, 135, 138, 247, and 253) out of 296 residues, but these enzymes exhibit different enzymatic properties ( Fig.…”

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confidence: 99%

Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205

et al. 2013

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bThe enzymes LinB UT and LinB MI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of ␤-hexachlorocyclohexane (␤-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB MI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB MI were categorized into three groups based on the efficiency of the first-step (from ␤-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB MI and its seven point mutants indicated how each mutated residue contributed to the first-and second-step conversions by LinB MI . The dynamics simulation analyses of wild-type LinB MI and LinB UT revealed that the entrance of the substrate access tunnel of LinB UT was more flexible than that of LinB MI , which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.H exachlorocyclohexane (HCH) is a six-chlorine-substituted cyclohexane. One of its isomers, the ␥ isomer, has insecticidal properties and has been widely used as an insecticide around the world (1). Although the use of ␥-HCH has been prohibited in most countries due to its toxicity and long persistence, the largescale production, widespread use, and dumping of the other noninsecticidal isomers (␣-, ␤-, and ␦-HCHs) in past decades still continue to create problems with HCH contamination in soil and groundwater (2). ␤-HCH in particular is a persistent and problematic isomer of HCH.Several ␤-HCH-degrading bacteria whose ␤-HCH-degrading enzymes can be utilized for bioremediation have been identified (3-5). LinB MI and LinB UT are haloalkane dehalogenases isolated from Sphingobium sp. MI1205 and Sphingobium japonicum UT26, respectively, that can cleave the carbon-halogen bond in ␤-HCH. Haloalkane dehalogenases belong to the ␣/␤-hydrolase family, and their catalytic mechanism consists of the following steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond in the substrate and formation of an intermediate covalently bound to the nucleophile, (iii) hydrolysis of the alkyl-enzyme intermediate, and (iv) release of halide ion and alcohol (6). LinB MI and LinB UT share 98% sequence identity, with only 7 different amino acid residues (at positions 81, 112, 134, 135, 138, 247, and 253) out of 296 residues, but these enzymes exhibit different enzymatic properties (Fig. 1). LinB MI catalyzes the two-step dehalogenation and converts ␤-HCH to 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and further to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) (7) in the manner of LinB2 from Sphingomonas sp. BHC-A (8) and LinB from Sphingobium indicum B90A (9), whereas LinB UT catalyzes only the firs...

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“…The catalytic aspartic acid is then regenerated through nucleophilic attack at Asp108 upon activation of a water molecule by histidine 272 [78].…”

Section: 1)mentioning

confidence: 99%