Molecular cloning, modeling, and site-directed mutagenesis of type III polyketide synthase from Sargassum binderi (Phaeophyta).

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C(6)-C(14)) to produce tri- and tetraketide pyrones. Mutations at H(331) and N(364) caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His(227) and Leu(366) play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.

A flavo-diiron protein from Desulfovibrio vulgaris with oxidase and nitric oxide reductase activities. Evidence for an in vivo nitric oxide scavenging function.

A few members of a widespread class of bacterial and archaeal flavo-diiron proteins, dubbed FprAs, have been shown to function as either oxidases (dioxygen reductases) or scavenging nitric oxide reductases, but the questions of which of these functions dominates in vivo for a given FprA and whether all FprAs function as oxidases or nitric oxide reductases remain to be clarified. To address these questions, an FprA has been characterized from the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris. The gene encoding this D. vulgaris FprA lies downstream of an operon encoding superoxide reductase and rubredoxin, consistent with an O(2)-scavenging oxidase function for this FprA. The recombinant D. vulgaris FprA can indeed serve as the terminal component of an NADH oxidase. However, this oxidase turnover results in irreversible inactivation of the enzyme. On the other hand, the recombinant D. vulgaris FprA shows robust anaerobic nitric oxide reductase activity in vitro and also protects a nitric oxide-sensitive Escherichia coli strain against exposure to exogenous nitric oxide. It is, therefore, proposed that this D. vulgaris FprA functions as a scavenging nitric oxide reductase in vivo and that this activity protects D. vulgaris against anaerobic exposure to nitric oxide. The location of a gene encoding a second FprA homologue in the D. vulgaris genome also suggests its involvement in nitrogen oxide metabolism.