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Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates
Author(s) -
Samanta Sudip K.,
Harwood Caroline S.
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04452.x
Subject(s) - biology , rhodopseudomonas palustris , dna ligase , biochemistry , enzyme kinetics , strain (injury) , nucleic acid sequence , enzyme , serine , nucleotide , peptide sequence , amino acid , coenzyme a , gene , active site , bacteria , genetics , anatomy , reductase
Summary Rhodopseudomonas palustris strain RCB100 degrades 3‐chlorobenzoate (3‐CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3‐CBA and determined its N‐terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate‐CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3‐CBA as a sole carbon source. The aliA gene from the 3‐CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His‐tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3‐CBA degrading strain was 10‐fold more active with 3‐CBA ( k cat / K m of 4.3 × 10 4 M −1 s −1 ) than the enzyme from the sequenced strain ( k cat / K m 0.32 × 10 4 M −1 s −1 ). The CGA009 enzyme was not sufficiently active with 3‐CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.