Adenosine kinase from Cryptosporidium parvum

Abstract

Analysis of the Cryptosporidium parvum genome demonstrates that the parasite cannot synthesize purines de novo and reveals that the sole route for purine salvage by the parasite is via adenosine kinase (CpAK). In order to initiate a biochemical characterization of CpAK and ultimately validate this apparently essential enzyme as a therapeutic target, the CpAK gene was redesigned for optimum codon usage, overexpressed in Escherichia coli, and the recombinant protein purified to homogeneity and characterized. CpAK appears to be specific for adenosine among the naturally occurring nucleosides but can utilize ATP, GTP, UTP and CTP as the phosphate donor. The enzyme exhibits K_m values of 1.4 μM for adenosine and 41 μM for ATP, has a pH optimum ∼7.0, and is dependent upon the presence of a divalent cation. Structure–activity data intimate that catalysis requires contacts between residues on CpAK with the six-position of the purine ring and the O2′ and O3′ hydroxyls of the ribose sugar. Additionally, 4-nitro-6-benzylthioinosine, a compound that demonstrates therapeutic promise against the related parasite Toxoplasma gondii, also inhibits adenosine phosphorylation by CpAK. The overproduction and purification of CpAK now enables a thorough evaluation of its potential as a drug target.

Introduction

Cryptosporidium parvum is the causative agent of cryptosporidiosis, an enteric disease afflicting millions of people worldwide [1]. C. parvum is present in 65–97% of surface waters [2], [3], [4], and cryptosporidiosis can be contracted through ingestion of as few as 30 infectious oocysts [5]. Because of the threat to public water supplies, C. parvum is listed as a Category B Biodefense Pathogen by the National Institutes of Health. In individuals with an intact immune system, cryptosporidiosis is probably mostly asymptomatic [6] but can present itself as an acute, self-limiting diarrhoea [7]. However, symptoms can be severe, unremitting, and life threatening among immunocompromised individuals [7], [8]. A 1993 outbreak of cryptosporidiosis in Milwaukee caused by contaminated drinking water resulted in ∼400,000 cases of gastrointestinal disease [9] and 54 deaths [10]. Unfortunately, there are no fully effective chemotherapeutic treatments currently available for treating or preventing cryptosporidiosis, especially in AIDS patients [11].

Rational and selective chemotherapies for any infectious disease require the exploitation of fundamental biochemical or metabolic discrepancies between pathogen and host. Unfortunately, the identification of drug targets in C. parvum has been hampered by minimal knowledge of the basic biochemistry of the parasite, mostly because of the lack of a continuous in vitro propagation system. The completion of the C. parvum genome sequence [12] has finally allowed insight into some of these biochemical pathways, and in silico metabolic reconstruction has revealed adaptation to extreme parasitism; C. parvum lacks a Krebs cycle, much of the oxidative phosphorylation pathway, and is incapable of synthesizing amino acids, purines, and pyrimidines de novo [12], [13].

The nutritional necessity for purines must be overcome through the obligatory salvage of host purines. The purine salvage pathway of C. parvum, compared to other protozoan parasites [6], is austere. The solitary route by which preformed host purines can be salvaged into the parasite nucleotide pool is via adenosine kinase (AK) [13]. AK (ATP: adenosine 5′-phosphotransferase, EC 2.7.1.20) catalyzes the ATP-dependent phosphorylation of the 5′-hydroxyl moiety of adenosine to form AMP and ADP [14], [15], [16]. The AMP product of the AK reaction can then be converted to guanylate nucleotides via the purine interconversion enzymes, AMP deaminase, IMP dehydrogenase, and GMP synthase (http://www.cryptodb.org). The C. parvum AK (CpAK) gene has been expressed in Toxoplasma gondii, a related apicomplexan parasite, and shown to encode a functional AK activity [13]. However, this heterologous expression system is not amenable to extensive biochemical investigations on the CpAK enzyme.

To overcome this limitation, a codon-optimized version of CpAK was synthesized and transformed into Escherichia coli. The CpAK protein was purified to homogeneity, and its basic kinetic properties are now reported. The properties of CpAK and its critical nutritional role in purine salvage by the parasite support further therapeutic validation of CpAK.