Kinetic and sequence-structure-function analysis of known LinA variants with different hexachlorocyclohexane isomers

• Published in: PloS one Vol. 6; no. 9; p. e25128
• Main Authors: Sharma, Pooja, Pandey, Rinku, Kumari, Kirti, Pandey, Gunjan, Jackson, Colin J, Russell, Robyn J, Oakeshott, John G, Lal, Rup
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.09.2011; Public Library of Science (PLoS)
• Subjects: Anticonvulsants - chemistry; Anticonvulsants - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biodegradation, Environmental; Biology; Bioremediation; Chromatography; Cloning; Coding; E coli; Ecosystems; Enzymes; Escherichia coli - enzymology; Function analysis; Genetic Variation - genetics; Hexachlorocyclohexane; Hexachlorocyclohexane - chemistry; Hexachlorocyclohexane - metabolism; Isomers; Kinetics; Laboratories; Lyases - genetics; Lyases - metabolism; Models, Chemical; Mutagenesis; Mutation; Pollutants; Proteins; Pseudomonas; Sphingomonas; Stereoisomerism; Stereoselectivity; Stereospecificity; Structure-function relationships; Studies; Trends; Zoology; Canberra Australian Capital Territory Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0025128

Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (-)-α -HCH are also discussed. Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2(B90A) A110T, A111C, A110T/A111C and LinA1(B90A) were constructed using the FoldX computer algorithm. Turnover rates (min(-1)) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA's, followed by the γ and then δ isomer. The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.