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

Abstract

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...

Kinetic and Sequence-Structure-Function Analysis of LinB Enzyme Variants with β- and δ-Hexachlorocyclohexane

“…As expected from previous reports, LinB UT26 yielded lower activities with either isomer [7], [13], [14], as did the closely related LinB SP+ . The other eight variants all showed 10–20 fold greater activities with β- than δ-HCH.…”

“…Notably, this variant yields a significantly lower specificity constant for β-HCH (0.02 mM −1 s −1 ) as compared to another relatively well characterized LinB, namely, LinB B90A (identical to LinB MI1205 , and LinB BHC-A and LinB pLB1 ) from Sphingobium indicum strain B90A (0.20 mM −1 s −1 ) [13]. Nonetheless the activity of LinB B90A for β-HCH is much lower than that of LinB UT26A for some of the other halogenated aliphatic compounds mentioned above (see [7], [13] for a comprehensive list of kinetic constants).…”

“…The best characterized LinB, LinB UT26A from Sphingobium japonicum strain UT26, has a broad substrate specificity, mainly due to a large active site volume, which includes monochloroalkanes (C3–C10), dichloroalkanes, bromoalkanes and chlorinated aliphatic alcohols [7], [10]. Notably, this variant yields a significantly lower specificity constant for β-HCH (0.02 mM −1 s −1 ) as compared to another relatively well characterized LinB, namely, LinB B90A (identical to LinB MI1205 , and LinB BHC-A and LinB pLB1 ) from Sphingobium indicum strain B90A (0.20 mM −1 s −1 ) [13]. Nonetheless the activity of LinB B90A for β-HCH is much lower than that of LinB UT26A for some of the other halogenated aliphatic compounds mentioned above (see [7], [13] for a comprehensive list of kinetic constants).…”

Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases

“…More divergent α/β-hydrolase-fold enzymes (only 10–20% aa identify) found in various bacteria or animals are C–C hydrolases (e.g., BphD from Burkholderia cepacia 44 ), epoxide ring hydrolases (e.g., human soluble epoxide hydrolase 67–69 ), and haloalkane hydrolases (e.g., LinB from Sphingobium sp. 70 ). These are too distantly related to be included in the tree in Figure S1.…”

Lessons from the genomes of lindane‐degrading sphingomonads

“…Functional analyses of LinB variants have also been done, especially with respect to β‐HCH degradation activity (Okai et al ., ). As mentioned previously, β‐HCH is the most recalcitrant among the four major isomers of t‐HCH (Willett et al ., ).…”