Lipases Provide a New Mechanistic Model for Polyhydroxybutyrate (PHB) Synthases:  Characterization of the Functional Residues in Chromatium vinosum PHB Synthase

Abstract

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

“…2). This architecture is reminiscent of that seen in lipases, as had been suggested based on sequence similarity and threading models (9,10,24,25) (Table 2). Structural comparison of the CnPhaC catalytic domain with these lipases reveals high structural similarity in the ␤-sheet core with variations in the lengths and relative placements of the surrounding ␣-helices (Fig.…”

“…1) (8,10,13,17). Asp 480 has been suggested to deprotonate the hydroxyl group of the second and subsequent HB-CoAs, allowing for ester formation and elongation of the PHB chain (8,9,18). Our structure of the catalytic domain of CnPhaC reveals these residues arranged together in a cavity that is ϳ10 Å from the nearest surface of the protein, defining the location of the active site (Fig.…”

“…Given the simi- NOVEMBER 25, 2016 • VOLUME 291 • NUMBER 48 larity in the standard pK a values of histidine and cysteine (6 and 8, respectively), modulation of the His 508 basicity through the Asp-His interaction is, in principle, unnecessary for this step. However, mutation of Asp 480 to Asn in CnPhaC was shown to result in a 50-fold decrease in the rate of enzyme acylation, suggesting that Asp 480 could play a role in nucleophilic activation, although mutation of the equivalent residue in the class III synthase from A. vinosum had no effect on the acylation rate (8,9). We note that it is also possible that the resting state of the enzyme is zwitterionic, containing a preformed cysteine thiolate and histidine imidazolium pair, as has been demonstrated in cysteine proteases (40,41).…”

“…The synthases are divided into four classes depending on their substrate specificity and subunit composition, although all classes are thought to have a common catalytic mechanism and a conserved active site architecture reminiscent of that seen in lipases (7)(8)(9)(10). The class I and class III synthases are the best studied and share a common substrate specificity for 3-(R)-hydroxybutyryl-CoA (HB-CoA) to form polyhydroxybutyrate (PHB) of high molecular mass (ϳ1-2 MDa) and low polydispersity (1,2,8,11,12).…”

“…The second, currently favored, mechanism (Fig. 1B) uses a single active site and requires both covalent and noncovalent intermediates during the catalytic cycle (9,10,20,21).…”