The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: Reaction mechanism and molecular evolution

Pavlová, Martina; Klvaňa, Martin; Jesenská, Andrea; Prokop, Zbyněk; Konečná, Hana; Sato, Takashi; Tsuda, Masashi; Nagata, Yasunobu; Damborský, Jiřı́

doi:10.1016/j.jsb.2006.09.004

Cited by 14 publications

(6 citation statements)

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“…3A). The core domain (residues 2 to 132 and 214 to 295) had a typical ␣/␤-hydrolase fold, as seen in other haloalkane dehalogenases (25)(26)(27)(28)(29). Unlike the core domain, the cap domain varied in the number and orientations of helices among haloalkane dehalogenases, and the cap domain (residues 133 to 213) of LinB MI was composed of four 3 10 and six ␣-helices.…”

Section: Resultsmentioning

confidence: 95%

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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Section: Resultsmentioning

confidence: 95%

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

et al. 2013

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“…HLDs represent one of the best characterized enzyme families that act on halogenated hydrocarbons and their derivatives [25]. During the past twenty five years, 17 different bacterial HLDs have been identified and at least partly biochemically characterized [26–38] (Prudnikova et al, in preparation). Characteristics of HLDs are summarized in Figure 2.…”

Section: Properties Of Hldsmentioning

confidence: 99%

“… Biochemical and structural properties of HLDs. ( A ) Summary of properties of DatA [35], DbeA [46] (Prudnikova et al, in preparation), DbjA [31, 45–47, 58], DhaA [27, 45–47, 49, 50, 55, 119, 120], DhlA [26, 46, 48, 50, 56, 121], DhmA [29, 32], DmbA [30, 46, 122], DmbB [30], DmbC [33, 46], DmlA [31, 47], DmmA [37], DpcA [38], DppA [36], DrbA [33, 46], LinB [45–47, 51, 123]. The isoelectric point ( pI ) and molecular weight (MW) were predicted by Expasy server [124].…”

Section: Properties Of Hldsmentioning

confidence: 99%

“…Although the expression and function of other HLDs in their original strains has not been identified, most HLDs can be heterologously expressed in Escherichia coli (Fig. 2) [26–39, 42] (Prudnikova et al in preparation). Protein yields obtainable from shake flask cultivations under laboratory conditions range between 1–150 mg L –1 , depending on the particular heterologous enzyme expressed and expression system used.…”

Section: Properties Of Hldsmentioning

confidence: 99%

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Haloalkane dehalogenases: Biotechnological applications

Koudeláková

Bidmanová

Dvořák

et al. 2012

Biotechnology Journal

138

120

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Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic preparation of optically pure building-blocks for organic synthesis; (ii) recycling of by-products from chemical processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein analysis. This review discusses the application of HLDs in the context of the biochemical properties of individual enzymes. Further extension of HLD uses within the field of biotechnology will require currently limiting factors - such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of organic co-solvents - to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solutions.

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“…A major deviation in the catalytic groups of HLDs and other non-hydrolytic enzymes such as the 4-chlorobenzoyl-coenzyme A dehalogenase is seen with the essential tryptophan residues in which two Trp 175,125 residues form a binding pocket for the incipient halide ion [ 19 ]. Initially, carboxylate oxygen of the aspartate launches a nucleophilic attack on the carbon atom of the substrate that is bonded with halogen [ 14 , 18 , 19 , 20 , 21 , 61 , 69 , 70 ]. It can be clearly seen from that the aspartate acts as nucleophile and histidine as base for different type of haloalkane dehalogenases but the acid can be glutamate or aspartate and the two halide-stabilizing residues can be any combination formed by tryptophan and asparagine mostly.…”

Section: Introductionmentioning

confidence: 99%

Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications

et al. 2018

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The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases—key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

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The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: Reaction mechanism and molecular evolution

Cited by 14 publications

References 73 publications

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

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

Haloalkane dehalogenases: Biotechnological applications

Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications

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