A Catalytic Mechanism Revealed by the Crystal Structures of the Imidazolonepropionase from Bacillus subtilis

Jun-Min Quan,Yu-He Liang,Xiao-Dong Su,Yamei Yu,Yu-Hui Dong,Santosh Panjikar,Erik Brostromer

doi:10.1074/jbc.m607703200

Abstract

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-l-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.

Highlights

The histidine degradation pathway is highly conserved from prokaryotes to eukaryotes [1, 2]
The histidine degradation pathway is operated by the hut genes, and the Bacillus subtilis hut operon encodes proteins in the order of HutP, HutH, HutU, HutI, HutG, HutM [3,4,5,6]
Crystal Structure of B. subtilis Imidazolonepropionase barrel and belonged to a novel ␣/␤ barrel amidohydrolase superfamily based on the residue-by-residue optimal alignment and superimposition of the three-dimensional structures of urease, phosphotriesterase, and adenosine deaminase [13]

Summary

Introduction

The histidine degradation pathway is highly conserved from prokaryotes to eukaryotes [1, 2]. We present the crystal structures of an imidazolonepropionase or HutI from B. subtilis, a native enzyme and an enzyme complex with its substrate analog imidazole-4-acetic acid (Fig. 1B), each to 2.0-Å resolution.

Results

Conclusion