Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile.

Laura Díaz-Sáez,William N. Hunter,Martin Zoltner,Velupillai Srikannathasan

doi:10.1042/bj20140511

Abstract

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+-dependent enzymes, there is little scope to increase co-ordination number during catalysis. We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.

Highlights

Tryptophan catabolism via the kynurenine pathway supports the biosynthesis of NAD+, important neuroactive intermediates in eukaryotes, anthranilate, quinolate and antibiotics in prokaryotes [1,2,3,4]
Bacillus anthracis KynB (BaKynB) was incubated with 8 mM L-kynurenine for 10 min at room temperature, and a structure was obtained from a crystal grown in a drop equilibrating with a reservoir containing 100 mM Tris/HCl, pH 8.5, 150 mM MgCl2, 30 % (w/v) PEG 4000 and 1.5 % (v/v) dioxane
During the final chromatography step of purification, i.e. sizeexclusion chromatography (SEC), all three proteins were eluted as a single species with approximate mass of 40 kDa and native gels identified that a single dimeric species was observed in each case

Summary

Introduction

Tryptophan catabolism via the kynurenine pathway supports the biosynthesis of NAD+ , important neuroactive intermediates in eukaryotes, anthranilate, quinolate and antibiotics in prokaryotes [1,2,3,4]. High-resolution crystal structures have been determined that allow us to describe the architecture of the enzyme extending to the details of the active site, to address metal ion identification and to investigate key features of molecular recognition at the catalytic centre.

Results

Conclusion