Role of an Essential Acyl Coenzyme A Carboxylase in the Primary and Secondary Metabolism of Streptomyces coelicolor A3(2)

Abstract

Two genes, accB and accE, that form part of the same operon, were cloned from Streptomyces coelicolor A3(2). AccB is homologous to the carboxyl transferase domain of several propionyl coezyme A (CoA) carboxylases and acyl-CoA carboxylases (ACCases) of actinomycete origin, while AccE shows no significant homology to any known protein. Expression of accB and accE in Escherichia coli and subsequent in vitro reconstitution of enzyme activity in the presence of the biotinylated protein AccA1 or AccA2 confirmed that AccB was the carboxyl transferase subunit of an ACCase. The additional presence of AccE considerably enhanced the activity of the enzyme complex, suggesting that this small polypeptide is a functional component of the ACCase. The impossibility of obtaining an accB null mutant and the thiostrepton growth dependency of a tipAp accB conditional mutant confirmed that AccB is essential for S. coelicolor viability. Normal growth phenotype in the absence of the inducer was restored in the conditional mutant by the addition of exogenous long-chain fatty acids in the medium, indicating that the inducer-dependent phenotype was specifically related to a conditional block in fatty acid biosynthesis. Thus, AccB, together with AccA2, which is also an essential protein (E. Rodriguez and H. Gramajo, Microbiology 143:3109-3119, 1999), are the most likely components of an ACCase whose main physiological role is the synthesis of malonyl-CoA, the first committed step of fatty acid synthesis. Although normal growth of the conditional mutant was restored by fatty acids, the cultures did not produce actinorhodin or undecylprodigiosin, suggesting a direct participation of this enzyme complex in the supply of malonyl-CoA for the synthesis of these secondary metabolites.

Kinetic and Structural Analysis of a New Group of Acyl-CoA Carboxylases Found in Streptomyces coelicolor A3(2)

“…In this paper we describe the kinetic properties of the two acyl-CoA carboxylases, ACC and PCC, previously described by our group (14,15). We also present data that address the functional significance of the ⑀ subunit in both enzyme complexes and propose a new group of enzyme within the acyl-CoA carboxylases.…”

“…Coomassie Brilliant Blue was used to stain protein bands. The biotinylated proteins were (15) detected by modification of Western blotting procedure described by Nikolau et al (25). After electrophoretic separation, proteins were electroblotted onto nitrocellulose membranes (Bio-Rad) and probed with alkaline phosphatase-streptavidin conjugate (AP-streptavidin diluted 1:10,000) (Bio-Rad).…”

“…In Streptomyces we were able to demonstrate, by constructing a conditional mutant in its carboxyltransferase component, AccB, that the main physiological role of the ACC complex was that of an ACC that synthesizes malonyl-CoA for both fatty acid and polyketide biosynthesis (15). Because polyketide synthetases are large multienzyme systems that are responsible for the stepwise biosynthesis of complex natural products from simple 2-, 3-, and 4-carbon building blocks such as acetyl-, propionyl-, and butyryl-CoA and their activated derivatives malonyl-, methylmalonyl-, and ethylmalonyl-CoA (17,18), it is obvious that the acyl-CoA carboxylases will certainly play a fundamental role for the biosynthesis of this pharmacologically important compounds (15).…”

“…This enzyme appeared to be formed by two subunits: the biotinylated ␣ subunit, encoded by either of the two identical genes named accA1 and accA2, and the carboxyltransferase component, encoded by pccB. The third acyl-CoA carboxylase characterized was shown to be essential for S. coelicolor viability, and it was called ACC (15). An interesting characteristic of this complex was that a third subunit (⑀), AccE, considerably enhanced the basal activity obtained by the ␣ and ␤ subunits, encoded by AccA2 and AccB, respectively.…”

“…This enzyme consisted of a biotinylated protein, PccA, of 88 kDa, and a non-biotinylated component, the carboxyltransferase, of 66 kDa. In our laboratory, two new carboxylases from S. coelicolor have been partially characterized at both the genetic and biochemical levels (14,15). One of them was defined as a PCC, because a mutation in its carboxyltransferase component, pccB, affected only PCC activity levels (14).…”