Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB.
The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.
Ma
Suzanne M
SM
Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.
Tang
Yi
Y
D000213:Acyl Carrier Protein / Q000737:chemistry; D051080:Acyl-Carrier Protein S-Malonyltransferase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D020134:Catalytic Domain / Q000502:physiology; D008969:Molecular Sequence Data; D048630:Polyketide Synthases / Q000737:chemistry*
D000213:Acyl Carrier Protein; D048630:Polyketide Synthases; D051080:Acyl-Carrier Protein S-Malonyltransferase