Exploring the Structure and Activity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26: Evidence for Product- and Water-Mediated Inhibition^,

Abstract

The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for example, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

“…The major catalytic domain of LinB belongs to the large and well-characterized ␣/␤-hydrolase fold superfamily of proteins, which characteristically carry out two-step hydrolytic reactions driven by a nucleophile (Asp) that is part of a catalytic triad and using an oxanion hole to stabilize the intermediate (69,114,117). LinB has been the subject of several crystallographic (92,123,166) and computational studies (32,33,69,122,130).…”

Structure and activity of DmmA, a marine haloalkane dehalogenase

“…This sequence-location difference of the catalytic pentad residues defines three evolutionary subfamilies of HLDs. 10 Two structures are known from evolutionary subfamily I (DhlA [11][12][13] and DppA 14 ), and four from subfamily II (DhaA, 15,16 LinB, [17][18][19][20] DbjA, 21 and Rv2578 22 ). Several structures include bound substrates, products, or covalent intermediates, which together with many kinetic studies, led to a detailed reaction mechanism for this enzyme class.…”

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

“…However, the activities of these mutants were still higher than that of LinB UT in both the first-and second-step dehalogenations, which suggested that one or more of the other five residues (T81, V112, T135, L138, and I253) uniquely found in LinB MI were also important for the high dehalogenation activity of LinB MI . To date, the crystal structure of LinB UT has been described (11)(12)(13)(14), whereas the crystal structure of LinB MI has not. To investigate how the seven residues that are different between LinB MI and LinB UT contribute to their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB MI and its seven point mutants, where each LinB MI -specific residue is mutated to the LinB UT -type residue (T81A, V112A, V134I, T135A, L138I, H247A, and I253M).…”