Molecular analysis of the role of tyrosine 224 in the active site of Streptomyces coelicolor RppA, a bacterial type III polyketide synthase.
Streptomyces coelicolor RppA (Sc-RppA), a bacterial type III polyketide synthase, utilizes malonyl-CoA as both starter and extender unit substrate to form 1,3,6,8-tetrahydroxynaphthalene (THN) (therefore RppA is also known as THN synthase (THNS)). The significance of the active site Tyr(224) for substrate specificity has been established previously, and its aromatic ring is believed to be essential for RppA to select malonyl-CoA as starter unit. Herein, we describe a series of Tyr(224) mutants of Sc-RppA including Y224F, Y224L, Y224C, Y224M, and Y224A that were able to catalyze a physiological assembly of THN, albeit with lower efficiency, challenging the necessity for the Tyr(224) aromatic ring. Steady-state kinetics and radioactive substrate binding analysis of the mutant enzymes corroborated these unexpected results. Functional examination of the Tyr(224) series of RppA mutants using diverse unnatural acyl-CoA substrates revealed the unique role of malonyl-CoA as starter unit substrate for RppA, leading to the development of a novel stericelectronic constraint model.
Li
Shengying
S
Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Grüschow
Sabine
S
Dordick
Jonathan S
JS
Sherman
David H
DH
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008316:Malonyl Coenzyme A / Q000378:metabolism; D009284:Naphthols / Q000378:metabolism; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000201:enzymology*; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
C121288:1,3,6,8-tetrahydroxynaphthalene; D001426:Bacterial Proteins; D009284:Naphthols; C412036:RppA protein, Synechocystis; D014443:Tyrosine; D008316:Malonyl Coenzyme A; D048630:Polyketide Synthases