Structure‐directed construction of a high‐performance version of the enzyme FabG from the photosynthetic microorganismSynechocystis sp. PCC 6803

Abstract

a b s t r a c tPhaB (acetoacetyl-CoA reductase) catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA in polyhydroxybutyrate (PHB) synthesis and FabG (3-ketoacyl-acyl-carrierprotein reductase) catalyzes the b-ketoacyl-ACP to yield (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis. Both of them have been classified into the same group EC 1.1.1. PhaB is limited with substrate specificities, while FabG was considered as a potential PhaB due to broad substrate selectivity despite of low activity. Here, X-ray crystal structures of FabG and PhaB from the photosynthetic microorganism Synechocystis sp. PCC 6803 were resolved. Based on them, a highperformance FabG on acyl-CoA directed by structural evolution was constructed that may serve as a critical enzyme to partition carbon flow from fatty acid synthesis to PHA.

FabG can function as PhaB for poly-3-hydroxybutyrate biosynthesis in photosynthetic cyanobacteria Synechocystis sp. PCC 6803

“…PCC 6803 (SpFabG and SpPhaB, respectively) were very similar, and SpFabG could act as a b-ketoacyl-CoA reductase in vitro. 11 These results implied that SpFabG might play a role in PHB synthesis and could participate in carbon flux distribution between FA and PHB in Synechocystis sp. PCC 6803.…”

“…These results demonstrated that SpFabG could complement PhaB when phaB was knocked out, although it could not work as efficiently as PhaB. It was reported that the enzyme activity of SpFabG using acetoacetyl-CoA as the substrate was 16% of that of SpPhaB in vitro, 11 which could account for the lower levels of PHB in the OvfabG C 4phaB mutant. However, SpFabG did not catalyze acetoacetyl-CoA when phaB was present, since overexpressing (OvfabG) or knocking down (fabG::C.K2) fabG had little impact on PHB content (Fig.…”

“…Enzyme activity assay and kinetic characterization of SpFabG using the substrate acetoacetyl-CoA SpFabG was expressed and purified as described previously. 11 The fractions containing SpFabG were concentrated using a 10 kDa Amicon concentrator (Millipore) and stored at ¡80 C until further use. The enzyme activity of SpFabG was determined as described previously.…”

“…The enzyme activity of SpFabG was determined as described previously. 11 Before the reaction, the protein solution was diluted to 0.1 mg/ml using buffer A (50 mM Tris-HCl pH 7.8, 300 mM NaCl, 1 mM EDTA, 5% glycerol (v/v), and 2 mM b-mercaptoethanol), and protein concentrations were determined using the Bradford assay with BSA as the standard. For optimum temperature determination, a mixture containing 200 mM acetoacetyl-CoA and 200 mM NADPH in 0.1 M PBS buffer (pH 7.0) was prepared in a volume of 100 mL at different temperatures ranging from 20 C to 60 C. The mixtures were preincubated for 5 min, and the reaction was initiated by the addition of 2 mL of diluted protein solution containing 0.2 mg SpFabG.…”

“…The only difference between them is the thioester bond that connects to ACP and CoA. 11 Therefore, the similarities in the substrates of FabG and PhaB could make it possible for FabG to participate in the synthesis of PHB and become the bridge between fatty acid metabolism and the PHB synthesis pathway. In vitro characterization showed that FabGs from some bacteria such as Pseudomonas and Escherichia coli could reversibly convert b-ketoacyl-CoA to D-b-hydroxyacyl-CoA.…”