Knowledge Bank

The metabolically diverse bacterium, Rhodobacter sphaeroides, was employed to study the assimilation of 3-hydroxypropionate during photoheterotrophic growth, specifically to test the hypothesis that 3-hydroxypropionate is activated via addition of a coenzyme A (CoA) thiol. One class of enzymes postulated to be responsible for catalyzing this reaction is AMP-forming synthetases, which hydrolyze ATP to add a free CoA to the target molecule. Several genes, rsp_0579 (acs) and rsp_1592 (pcst), with the potential to encode a 3-hydroxypropionyl-CoA synthetase were bioinformatically identified in the genome of R. sphaeroides. The purpose of this study was to characterize an annotated propionyl-CoA synthetase (Pcst) using a genetic and biochemical approach in order to determine its role in 3-hydroxypropionate assimilation. An in- frame pcst deletion mutant of R. sphaeroides (Δpcst) was generated; increased doubling time of Δpcst was observed during growth with 3-hydroxy propionate as the sole carbon source. Additionally, a decrease in CoA synthetase-specific activity towards acetate, propionate, and 3- hydroxypropionate was detected in the cell extract of Δpcst. A CoA synthetase-specific substrate dependence assays of heterologously produced and purified His6-tagged Pcst show a 100-fold higher catalytic efficiency of Pcst with propionate in comparison to acetate and 3-hydroxy- propionate. Pcst was characterized as a propionyl-CoA synthetase that displayed lower activity with substrates acetate and 3-hydroxypropionate. Pcst appears to contribute to assimilation of 3- hydroxypropionate in vivo, although not as the sole enzyme.