Enzymatic Extender Unit Generation for In Vitro Polyketide Synthase Reactions: Structural and Func-tional Showcasing of Streptomyces coelicolor MatB

Abstract

In vitro experiments with modular polyketide synthases (PKSs) are often limited by the availability of polyketide extender units. To determine the polyketide extender units that can be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in vitro PKS studies. Two ternary product complex structures, one containing malonyl-CoA and AMP and the other containing (2R)-methylmalonyl-CoA and AMP, were solved to 1.45 Å and 1.43 Å resolution, respectively. MatB crystallized in the thioester-forming conformation, making extensive interactions with the bound extender unit products. This first structural characterization of an adenylate-forming enzyme that activates diacids reveals the molecular details for how malonate and its derivatives are accepted. The orientation of the α-methyl group of bound (2R)-methylmalonyl-CoA, indicates that it is necessary to epimerize α-substituted extender units formed by MatB before they can be accepted by PKS acyltransferase domains. We demonstrate the in vitro incorporation of methylmalonyl groups ligated by MatB to CoA, pantetheine, or N-acetylcysteamine into a triketide pyrone by the terminal module of the 6-deoxyerythronolide B synthase. Additionally, a means for quantitatively monitoring certain in vitro PKS reactions using MatB is presented.

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

“…Since our study was completed, the crystal structure (Protein Data Bank code 3NYR) was published for a previously uncharacterized malonyl-CoA synthetase from Streptomyces coelicolor (38). This enzyme is active toward malonate, methylmalonate, ethylmalonate, methoxymalonate, and hydroxymalonate and exhibits 37% sequence identity to the human ACSF3 enzyme.…”

Structure-Guided Expansion of the Substrate Range of Methylmalonyl Coenzyme A Synthetase (MatB) of Rhodopseudomonas palustris

“…However, RpMatB is 38% identical and 53% similar to MatB from Streptomyces coelicolor (ScMatB), which utilizes malonate and methylmalonate as substrates and whose structure was recently solved in the thioester-forming conformation (27). Since the thioester-forming conformation is highly conserved within the acyl-CoA synthetase family, the ScMatB structure is an ideal template for modeling the RpMatB thioester-forming conformation.…”

“…However, RlMatB activity with methylmalonate is only ϳ20% of its activity with malonate (3,41,50). It was reported that Streptomyces coelicolor MatB (ScMatB) could synthesize 15 different polyketide extender units, encompassing all of the combinations of five different malonate derivatives and three different thiol acceptors (CoA, pantetheine, and N-acetylcysteamine), which could be used for in vitro polyketide synthesis (27), although the kinetics of this enzyme were not reported. This opened up the possibility that characterization of MatB homologues from different organisms may lead to expanded substrate specificity and improved activity for different malonate derivatives.…”

“…1) and has been isolated from a variety of soil bacteria and from mammalian mitochondria (27,33,34,63). The beststudied member of this group is MatB from the nitrogen-fixing legume symbiont Rhizobium leguminosarum bv.…”

“…In support of its biological role, RpMatB can efficiently use methylmalonate and has significant activity with ethylmalonate. To further expand the substrate specificity of RpMatB, we determined both the apo and ATP-bound crystal structures of RpMatB and utilized these two structures combined with a thioester-forming conformation structure of ScMatB (27) as a homology model to identify residue T208 in the binding pocket that influences the binding affinity of malonate derivatives. Two RpMatB variants, RpMatB T208A and RpMatB T208G , had increased activity with the larger malonate derivatives ethylmalonate and butylmalonate.…”