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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such as<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>often do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacterium<jats:named-content content-type="genus-species">Rhodopseudomonas palustris</jats:named-content>, MatB (<jats:italic>Rp</jats:italic>MatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report the<jats:italic>apo</jats:italic>(1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis of<jats:italic>Rp</jats:italic>MatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site of<jats:italic>Rp</jats:italic>MatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.</jats:p>

Structural Insights into the Substrate Specificity of the Rhodopseudomonas palustris Protein Acetyltransferase RpPat

“…Growth of R. palustris on Methyl Malonate Is Not under Acetylation Control-We recently showed that matB is required for photoheterotrophic growth of R. palustris on methyl malonate as a carbon source (36). If RpMatB activity were regulated by acetylation in vivo, we would expect that a deacetylase-deficient strain (⌬srtN ⌬ldaA) would have a growth defect, as is observed during growth on benzoate (Fig.…”

“…2H), it was unclear why this region was important for recognition or acetylation by RpPat. We hypothesized that there was a structural difference between wild-type RpMatB and its chimeras, and we attempted to crystallize both the RpMatB-RpPimA P9 and RpMatB-BxBclM B1 chimeras so that we could compare them with RpMatB and BxBclM, whose structures have been previously published (36,45). Attempts to crystallize either RpPimA or the RpMatB-RpPimA P9 chimera were not successful; therefore, only the RpMatB-BxBclM B1 chimera was pursued.…”

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

“…14D). It seems that the shape and electrostatic potential of the chimera loop play important roles, as minor changes in the loop allow an acyl-CoA synthetase to "escape" acetylation by RpPat (214). This indicates that although acetylation motifs may suggest that a protein is controlled by RLA, each substrate should be validated experimentally, as structural elements outside the motif can affect the ability of the acetyltransferase to recognize and acetylate the target.…”

“…The use of RLA to control acyl-CoA synthetases by acetylation or propionylation is not unique to Gammaproteobacteria, since there is abundant evidence of the same type of control happening in the alphaproteobacterium R. palustris. The common theme here is that RLA helps maintain a balance in the intracellular acetyl-CoA (or propionyl-CoA) pools, while an acyl-CoA synthetase (MatB) that contributes to the succinyl-CoA pool is not under RLA control (28,214). It is possible that acetylation of these AMP-forming acyl-CoA synthetases may control CoA homeostasis by preventing the depletion of CoA or buildup of acetylCoA or propionyl-CoA.…”

“…However, recent results support the conclusion that this motif is not sufficient for acetylation to occur. RpMatB, an acyl-CoA synthetase which activates the dicarboxylic acid methylmalonate to methylmalonyl-CoA (214), contains the PX 4 GK motif but is not acetylated by RpPat (215) (Fig. 14B).…”