Substrate specificity of haloalkane dehalogenases

Abstract

An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae

“…We observe a difference in substrate specificity between the highly similar HLD-II family enzymes, HanR and LinB [15]. LinB displays a significant drop of activity as the carbon chain becomes longer from the substrates 1-bromobutane to 1-bromoxehane.…”

“…The residues lining the substrate pocket mainly belong to the cap domain and define substrate specificity. Minor differences in the substrate pocket can dramatically affect the substrate specificity of very similar HLDs [15].…”

“…The division of HLDs into three phylogenetic subfamilies was defined by Chovancova et al in 2007 [14], depending on the amino acid content of the catalytic pentad. Classification of these HLDs into four groups based on substrate specificity revealed there was no clear correlation between substrate specificity and phylogenetic subfamilies [15]. More recently, the characterisation of a HLD from Agrobacterium tumefaciens that has a unique halide-stabilizing tyrosine residue (Tyr109) in place of the conserved Trp residue [16] has required an extension of the existing phylogenetic families.…”

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

“…Intriguingly, these SSG groupings do not significantly correlate with the phylogenetic subfamilies. 13 This disagreement underscores the need to characterize more HLDs to clarify the sequencestructure-function relationship within this industrially important family of enzymes. The first identified and best characterized HLD is DhlA, from Xanthobacter autotrophicus.…”

“…An alternative grouping of HLDs by substrate specificity profile produces four substrate specificity groups (SSG). 13 HLDs grouped in SSG-I display activity towards most of the tested substrates including poorly degraded compounds such as DCE and TCP. Those that are grouped in SSG-II show more restricted substrate specificity profile than SSG-I with lack of activity towards 1,3-diiodopropane.…”