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.
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 (kcat/Km of 4.3 x 10(4) M(-1) s(-1)) than the enzyme from the sequenced strain (kcat/Km 0.32 x 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.
Samanta
Sudip K
SK
Department of Microbiology, The University of Iowa, 3-450 BSB, Iowa City, IA 52242, USA.
Harwood
Caroline S
CS
D000214:Acyl Coenzyme A / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002723:Chlorobenzoates / Q000378:metabolism*; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016384:Consensus Sequence; D016680:Genome, Bacterial; D007700:Kinetics; D012241:Rhodopseudomonas / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D002723:Chlorobenzoates; C036427:3-chlorobenzoic acid; C046663:cyclohexanecarboxyl-coenzyme A; D003066:Coenzyme A Ligases; C500453:cyclohexanecarboxylate-CoA ligase