PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.

Overexpression, purification and crystallization of PhzA, the first enzyme of the phenazine biosynthesis pathway of Pseudomonas fluorescens 2-79.

Phenazines are broad-spectrum antibiotic metabolites produced by organisms such as Pseudomonas and Streptomyces. Phenazines have been shown to enhance microbial competitiveness and the pathogenic potential of the organisms that synthesize them. PhzA (163 residues, approximate molecular weight 18.7 kDa) is the product of the first of seven genes of the operon responsible for phenazine biosynthesis in P. fluorescens 2-79. This enzyme is thought to catalyse one of the final steps in the formation of phenazine-1-carboxylic acid, the end product of phenazine biosynthesis in P. fluorescens 2-79. Here, the purification and crystallization of recombinant PhzA are reported. Crystals diffracting to 2.1 angstroms were obtained using 1.6 M magnesium sulfate and 2-morpholinoethanesulfonic acid monohydrate (MES) buffer pH 5.2-5.6. Crystals of both native and seleno-L-methionine-labelled protein belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 66.8, b = 75.3, c = 84.5 angstroms. The asymmetric unit contains one dimer of PhzA.