Crystal Structures Representing the Michaelis Complex and the Thiouronium Reaction Intermediate of Pseudomonas aeruginosa Arginine Deiminase

Andrey Galkin,Xuefeng Lu,Debra Dunaway-Mariano,Osnat Herzberg

doi:10.1074/jbc.m505471200

Abstract

L-arginine deiminase (ADI) catalyzes the irreversible hydrolysis of L-arginine to citrulline and ammonia. In a previous report of the structure of apoADI from Pseudomonas aeruginosa, the four residues that form the catalytic motif were identified as Cys406, His278, Asp280, and Asp166. The function of Cys406 in nucleophilic catalysis has been demonstrated by transient kinetic studies. In this study, the structure of the C406A mutant in complex with L-arginine is reported to provide a snapshot of the enzyme.substrate complex. Through the comparison of the structures of apoenzyme and substrate-bound enzyme, a substrate-induced conformational transition, which might play an important role in activity regulation, was discovered. Furthermore, the position of the guanidinium group of the bound substrate relative to the side chains of His278, Asp280, and Asp166 indicated that these residues mediate multiple proton transfers. His278 and Asp280, which are positioned to activate the water nucleophile in the hydrolysis of the S-alkylthiouronium intermediate, were replaced with alanine to stabilize the intermediate for structure determination. The structures determined for the H278A and D280A mutants co-crystallized with L-arginine provide a snapshot of the S-alkylthiouronium adduct formed by attack of Cys406 on the guanidinium carbon of L-arginine followed by the elimination of ammonia. Asp280 and Asp166 engage in ionic interactions with the guanidinium group in the C406A ADI. L-arginine structure and might orient the reaction center and participate in proton transfer. Structure determination of D166A revealed the apoD166A ADI. The collection of structures is interpreted in the context of recent biochemical data to propose a model for ADI substrate recognition and catalysis.

Highlights

L-Arginine is used by a number of pathogenic microorganisms to generate ATP via the arginine dihydrolase pathway (1, 2)
Based on the structural similarity and conservation of several key residues in the active site of DDAH and Arginine deiminase (ADI), we proposed a model of arginine binding to ADI in which the guanidinium group is positioned in close proximity to the catalytic Cys[406] (5)
We focused on obtaining the structure of the PaADI1⁄7L-arginine complex so that we could identify conformational changes that occur upon substrate binding and determine the orientation of substrate binding and catalytic groups in the enzyme1⁄7substrate complex

Summary

Introduction

L-Arginine is used by a number of pathogenic microorganisms to generate ATP via the arginine dihydrolase pathway (1, 2). Based on the structural similarity and conservation of several key residues in the active site of DDAH and ADI, we proposed a model of arginine binding to ADI in which the guanidinium group is positioned in close proximity to the catalytic Cys[406] (5). The structural data suggested a nucleophilic attack by the thiol group of Cys[406] on the guanidinium carbon of the arginine substrate. The C406A-arginine structure guided the design of active site mutants in which substrate activation and/or general acid/base catalysis might be impaired. The H278A, D166A, and D180A mutants were prepared and subjected to kinetic analysis (the results of which are reported in a separate paper)[3] and to crystallization in the presence of L-arginine followed by x-ray structure determination. The structures reported in this paper, are interpreted in the context of the biochemical data to support a model for PaADI substrate recognition and catalysis. PROCHECK was used for analysis of geometry (15), QUANTA for molecular modeling and structural alignment (Molecular Simulations Inc.), and PYMOL for depiction of the structures (16)

Objectives

Methods

Results