Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis

Abstract

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

Crystal Structure of a Bacterial Type III Polyketide Synthase and Enzymatic Control of Reactive Polyketide Intermediates

“…Although previous plant PKS-based homology modeling failed to predict the many subtle backbone differences observed throughout this first bacterial type III PKS crystal structure reported here, there are no drastic rearrangements of the conserved ␣␤␣␤␣-fold or dimer interface in THNS. Our crystal structure shows that homology-based assignments of the THNS catalytic triad and other active site residues are essentially accurate, as previously supported by in vitro assays of Streptomyces griseus THNS point mutants (7).…”

“…Notably, these residues are conserved in all of the functionally confirmed bacterial THNS sequences discovered to date (5-8). Funa and co-workers recently identified the importance of Tyr 224 for THNS starter specificity (7). Although mutation to Phe or Trp was tolerated by THNS, nonconservative mutations at this position abrogated the enzymatic loading of the physiological malonyl starter.…”

“…Whereas type I PKSs and their fatty acid synthase ancestors have been intensely studied, their three-dimensional domain organization remains obscure due to their size and complexity. In stark contrast, bacteria use a structurally simple type III PKS architecturally organized as a homodimeric condensing enzyme (ϳ40 kDa) to synthesize THN from five coenzyme A (CoA)-tethered malonyl units, without the use of additional enzymes or catalytic domains (5)(6)(7)(8).…”

A New Family of Type III Polyketide Synthases in Mycobacterium tuberculosis

“…6A) have implicated its role in starter unit selectivity and determination of the chain length of the growing polyketide (13). Mutation of the analogous Ala 305 residue to a bulkier isoleucine residue in THNS protein abolished the synthesis of tetraketide pyrones, and only triketide pyrones were observed (18). In PKS18, Leu 348 was mutated to a smaller serine residue.…”

“…Over the past several years, CHS homologues have also been reported in bacteria. The type III PKS involved in the biosynthesis of 1,3,6,8-tetrahydroxynaphthalene (THN) has now been characterized from S. griseus (16), S. erythraea (17), S. antibioticus (18) and S. coelicolor (19). All of these THN synthases (THNSs) use malonyl-CoA as starter and extender units.…”