Crystallization and structure elucidation of benzoic acid-specific type III polyketide synthases

Abstract

Benzophenone synthase (BPS) and biphenyl synthase (BIS) catalyze iterative condensation of benzoyl-CoA with three molecules of malonyl-CoA to form a linear tetraketide. This intermediate undergoes C6->C1 Claisen cyclization by BPS to form trihydroxybenzophenone, but C2->C7 aldol cyclization by BIS to form dihydroxybiphenyl. Crystal structures of a number of type III polyketides synthases (PKS) hypothesized explanations for the two different mechanisms of these enzymes, but it remains open if either electronic or steric factors are the major reasons for the alternative cyclizations. Protein expression and purification of the His6-tagged proteins were established using high culture volumes for Hypericum androsaemum BPS (HaBPS), its mutant HaBPST135L, H. sampsonii BPS (HsBPS), and Malus domestica BIS3 (MdBIS3). The crystal structures of HaBPS and HaBPST135L were solved at 2.1 A and 1.4 A, respectively. The smaller active site pocket in HaBPST135L explains the production of the short triketide chain. The crystal structure of MdBIS3 was solved at 1.9 A. It identified a new active site pocket, which resembled that of HaBPST135L and seems to be a characteristic pocket of benzoic acid-specific type III PKSs. Ala127 in MdBIS3 and Thr135 in HaBPS line this novel pocket. A specific water molecule was found in the new pocket of HaBPS and MdBIS3. The orientations of Thr135 and Gly166 of HaBPS influence the hydrogen bond network around the new water molecule. Soaking and co-crystallization were carried out using the active site mutants HaBPSC167A and MdBIS3C159A. Benzoyl-CoA was modeled in one of the datasets of soaked MdBIS3 crystals. The crystal structure of HsBPS was solved at 1.7 A. In comparison to HaBPS, it has many displaced loops, which surround the active site pocket and also enclose surface areas with amino acids differing between the two enzymes. Transformation of BPS into BIS can identify the main residues that are responsible for the different condensation and cyclization mechanisms. However, 14-fold and 13-fold mutants of BPS exhibited neither BIS nor BPS activity. The active site pockets of HaBPS and MdBIS3 demonstrate high similarities in their geometry with two important alkaloid-synthesizing plant type III PKSs, namely Citrus microcarpa ACS and C. microcarpa QNS. This will provide the chance of transforming by mutagenesis benzoic acid-specific type III PKSs into ACS and QNS.