The Conserved Active Site Asparagine in Class I Ribonucleotide Reductase Is Essential for Catalysis

Alex Kasrayan,Annika L Persson,Margareta Sahlin,Britt-Marie Sjöberg

doi:10.1074/jbc.m106538200

Abstract

The active site residue Asn-437 in protein R1 of the Escherichia coli ribonucleotide reductase makes a hydrogen bond to the 2'-OH group of the substrate. To elucidate its role(s) during catalysis, Asn-437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln). All mutant proteins were incapable of enzymatic turnover but promoted rapid protein R2 tyrosyl radical decay in the presence of the k(cat) inhibitor 2'-azido-2'-deoxy-CDP with similar decay rate constants as the wild-type R1. These results show that all Asn-437 mutants can perform 3'-H abstraction, the first substrate-related step in the reaction mechanism. The most interesting observation was that three of the mutant proteins (N437A/S/D) behaved as suicidal enzymes by catalyzing a rapid tyrosyl radical decay also in reaction mixtures containing the natural substrate CDP. The suicidal CDP-dependent reaction was interpreted to suggest elimination of the substrate's protonated 2'-OH group in the form of water, a step that has been proposed to drive the 3'-H abstraction step. A furanone-related chromophore was formed in the N437D reaction, which is indicative of stalling of the reaction mechanism at the reduction step. We conclude that Asn-437 is essential for catalysis but not for 3'-H abstraction. We propose that the suicidal N437A, N437S, and N437D mutants can also catalyze the water elimination step, whereas the inert N437Q mutant cannot. Our results suggest that Asn-437, apart from hydrogen bonding to the substrate, also participates in the reduction steps of catalysis by class I ribonucleotide reductase.

Highlights

The enzyme ribonucleotide reductase (RNR)1 catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides [1]
These results show that all Asn-437 mutants can perform 3؅-H abstraction, the first substrate-related step in the reaction mechanism
A furanone-related chromophore was formed in the N437D reaction, which is indicative of stalling of the reaction mechanism at the reduction step

Summary

Introduction

The enzyme ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides [1]. Three major RNR classes are known [1, 2] Despite their different subunit composition and metal and cofactor requirements, their substrate binding domains are homologous [3], and they are all considered to use radical chemistry involving a thiyl radical initiating catalysis [4, 5]. The mode of thiyl radical formation as well as the details of the reaction mechanism differ between the classes, and only two cysteines in the active site are fully conserved in all classes. This study shows that an asparagine that is conserved in the active sites of class I and II RNRs is essential for catalysis. All class I RNRs have an array of conserved hydrogen-bonded residues between the active site of R1 and the tyrosyl radical of R2 [6, 7, 15]. The suicidal mutant protein E441Q allowed trapping and identification of a disulfide anion radical intermediate [34, 35], the first demonstration of one of the postulated radical intermediates of the wild-type reaction mechanism

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