Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Abstract

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The L-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms.The enzyme is a homodimer with each monomer being composed of a core-domain of a -sheet bundle surrounded by -helices and an -helical sub-domain. This fold is similar to previously solved mesophilic L-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor L-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60ºC and a half-life of over 1 hour at 70ºC. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 hour in 20% v/v DMSO.3

Marine Rhodobacteraceae l‐haloacid dehalogenase contains a novel His/Glu dyad that could activate the catalytic water

“…In comparison, l ‐DEX YL and DehIVa have slightly lower K m values, of 1.1 m m and 1.13 m m , respectively . DehSft has a comparable K m value of 6.23 m m .…”

“…The structure of a d ‐lactic acid product bound in the active site of l ‐DEX YL has also been reported . The structure of l ‐lactic acid bound in the active site of DehSft is proposed to mimic the prereaction Michaelis complex. The general S N 2 nucleophilic substitution mechanism has been proposed based on the structures of the characterized l ‐HAD enzymes .…”

“…These include the l ‐HADs from Pseudomonas sp. strain YL ( l ‐DEX YL) , Xanthobacter autotrophicus GJ10 (DhlB) , Burkholderia cepacia MBA4 (DehIVa) and Sulfolobus tokodaii (DehSft) . The structures of the ester intermediates bound to the catalytic aspartate have been determined for l ‐DEX YL , DhlB and DehIVa .…”

Halophiles: biology, adaptation, and their role in decontamination of hypersaline environments

“…Over the past decade, a variety of thermophilic (Rye et al 2009), psychrophilic (Drienovska et al 2012;Novak et al 2013), and non-extremophilic microbes capable of degrading 2,4-D (Fulthorpe et al 1995;González et al 2012;Kumar et al 2014;Samir et al 2015) have been isolated from contaminated marine environments (Chiba et al 2009;Fulthorpe et al 1995;González et al 2012;Kumar et al 2014;Novak et al 2014;Samir et al 2015). However, studies on bioremediation of chlorinated compounds under hypersaline environments are sparse.…”

Sugar (ribose), spice (peroxidase) and all things nice (laccase hair‐dyes)

“…Some of these dehalogenases have enhanced activities and this appears to arise from their sequence diversity (less than 30% sequence identity for HADs) (Prudnikova et al. , 2009; Rye et al. , 2009).…”