Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

Highlights

• Nucleoside hydrolase (rihC) from Escherichia coli has been cloned, expressed, and purified to homogeneity.

• Substrate specificity and Michaelis constants have been determined for common nucleosides.

• Structure/activity has been studied using a number of nucleoside analogs.

Abstract

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.

Introduction

Nucleoside hydrolases are a class of enzymes that hydrolyze the N-glycosidic bond of selected nucleosides between the base and sugar. They have been isolated from a number of sources including bacteria [1], [2], [3], parasitic protozoans [4], [5], [6], [7], plants [8], [9], [10], marine invertebrates [11], and baker's yeast [12]. However, while nucleoside hydrolases are widely distributed, they have not been found in mammals [13].

In parasitic protozoans, the nucleoside hydrolases salvage purine ribonucleoside bases for recycling [14]. Being absent in mammals, but necessary to protozoans, they are attractive targets for drugs to treat diseases such as malaria and Chaga's disease [15]. In other organisms, such as prokaryotes and higher eukaryotes, the enzyme carries out a variety of species-specific roles [16], [17], [18]. The nucleoside hydrolases characterized to date are metalloproteins containing a Ca^2 + ion found within a group of aspartate residues (DXDXXXDD) located at the N-terminus [13], [19], [20].

The most extensively studied nucleoside hydrolase is inosine–uridine nucleoside hydrolase (IU-NH) isolated from Crithidia fasciculata. This enzyme, part of the purine salvage pathway, has been cloned and expressed in Escherichia coli [21]. The transition state has been determined using kinetic isotope effects [22]. The transition state for purine nucleosides includes an oxocarbenium ion, protonation of N7, and a C3'-exo conformation of the sugar. A series of inhibitors based on this transition state have been synthesized [23]. An X-ray crystal structure of IU-NH complexed with p-aminophenyl-(1S)-iminoribitol has been determined containing a calcium ion bound to a group of aspartate residues at the bottom of the active site and identifying His241 as a proton donor for activation of the purine leaving group [24].

Peterson and Moller have identified three nucleoside hydrolases in E. coli extracts designated rihA, rihB, and rihC [25]. The three enzymes differ in their substrate specificity, with rihA and rihB being pyrimidine-specific and rihC able to hydrolyze both purine and pyrimidine ribonucleosides. Since E. coli recycles nucleoside bases using nucleoside phosphorylase rather than nucleoside hydrolases, the metabolic role of the nucleoside hydrolases in E. coli is not known. Of the three identified nucleoside hydrolases from E. coli, two have known crystal structures, rihA, also known as ybeK, and rihB, previously known as yeiK [26], [27]. The transition state has been determined for RihC previously known as yaaF, the third nucleoside hydrolase [28]. The characteristics of the RihC transition state are similar to those of the transition state of IU-NH isolated from C. fasciculata.

Nucleoside hydrolases have traditionally been classified based on their substrate specificities into the purine-specific, the pyrimidine-specific, the 6-oxopurine-specific and the nonspecific [13]. Alternatively, the nucleoside hydrolases can be classified based on sequence similarity and active site residues [26]. In this scheme, Group I proteins contain a conserved {V,I,L,M}HD{P,A,L} tetrapeptide sequence approximately 230 amino acids from the N-terminal Ca^2 + ion binding segment. This group contains both pyrimidine-specific and nonspecific nucleoside hydrolases. Group II nucleoside hydrolases replace the essential histidine of the Group I nucleosides hydrolases with an aromatic residue such as tyrosine or tryptophan. Group III contain an XCDX sequence in which the catalytic His239 of Group I nucleoside hydrolases is replaced with a cysteine residue. Based on its sequence, RihC from E. coli belongs to the group I nucleoside hydrolases along with yeiK and ybeK from E. coli, URH1 from S. cerevisiae, IU-NH from L. major, and IU-NH from C. fasciculata.

We report here the expression and purification of a full-length clone of rihC, along with its substrate specificity, the equilibrium constant of the inosine formation reaction, and state of oligomerization.