Role of the active site cysteine of DpgA, a bacterial type III polyketide synthase.
DpgA is a bacterial type III polyketide synthase (PKS) that decarboxylates and condenses four malonyl-CoA molecules to produce 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) in the biosynthetic pathway to 3,5-dihydroxyphenylglycine, a key nonproteinogenic residue in the vancomycin family of antibiotics. DpgA has the conserved catalytic triad of Cys/His/Asn typical of type III PKS enzymes, and has been assumed to use Cys160 as the catalytic nucleophile to create a series of elongating acyl-S-enzyme intermediates prior to the C(8) to C(3) cyclization step. Incubation of purified DpgA with [(14)C]-malonyl-CoA followed by acid quench during turnover leads to accumulation of 10-15% of the DpgA molecules covalently acylated. Mutation of the active site Cys160 to Ala abrogated detectable covalent acylation, but the C160A mutant retained 50% of the V(max) for DPA-CoA formation, with a k(cat) still at 0.5 catalytic turnovers/min. For comparison, a C190A mutant retained wild-type activity, while the H296A mutant, in which the side chain of the presumed catalytic His is removed, had a 6-fold drop in k(cat). During turnover, purified DpgA produced 1.2 equivalents of acetyl-CoA for each DPA-CoA, indicating 23% uncoupled decarboxylation competing with condensative C-C coupling. The C160A mutant showed an increased partition ratio for malonyl-CoA decarboxylation to acetyl-CoA vs condensation to DPA-CoA, reflecting more uncoupling in the mutant enzyme. The Cys-to-Ala mutant thus shows the unexpected result that, when the normal acyl-S-enzyme mechanism for this type III PKS elongation/cyclization catalyst is removed, it can still carry out the regioselective construction of the eight-carbon DPA-CoA skeleton with surprising efficiency.
Tseng
Claire C
CC
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
McLoughlin
Shaun M
SM
Kelleher
Neil L
NL
Walsh
Christopher T
CT
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002250:Carbon Radioisotopes / Q000378:metabolism; D002384:Catalysis; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003653:Decarboxylation; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; C439058:DpgA protein, Amycolatopsis mediterranei; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D003066:Coenzyme A Ligases; D003545:Cysteine; D000409:Alanine