The Rv1712 Locus from Mycobacterium tuberculosis H37Rv Codes for a Functional CMP Kinase That Preferentially Phosphorylates dCMP

Abstract

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.Nucleoside monophosphate (NMP) kinases are key enzymes in the metabolism of ribo-and deoxyribonucleoside triphosphates, catalyzing the reversible phosphoryl transfer from a nucleoside triphosphate (usually ATP) to a specific NMP (25). The resulting nucleoside diphosphates are subsequently phosphorylated to generate nucleoside triphosphates, precursors of nucleic acids. Bacterial CMP kinase (CMK), which is part of pyrimidine nucleotide interconversion pathways, catalyzes the transfer of a phosphate group from ATP to either CMP or dCMP to form CDP or dCDP and ADP, respectively (1). In Mycobacterium tuberculosis, the causative agent of human tuberculosis, a putative cmk (Rv1712) gene has been identified in the genome of the H37Rv strain by sequence similarity (8).In addition, it has been proposed that the cmk gene product is essential for the optimal in vitro growth of M. tuberculosis based on transposon site hybridization studies (15). However, this approach is a large-scale screening methodology, and hence, cmk gene manipulation experiments must be carried out to firmly support the hypothesis of its essentiality in M. tuberculosis. The first step should thus be demonstration that the Rv1712 locus indeed encodes a protein having CMK activity in M. tuberculosis. Here, we report heterologous recombinant protein expression, purification to homogeneity, N-terminal amino acid sequencing, electrospray ionization-mass spectrometry (ESI-MS) analysis, and size exclusion chromatography of a functional cmk-encoded M. tuberculosis CMK (MtCMK). Steady-state kinetics showed that MtCMK preferentially phosphorylates CMP and dCMP, and double-reciprocal plots indicated a sequential mechanism for MtCMK with ternary complex formation. The availability of the MtCMK protein in large quantities will allow further functional and structural efforts to be undertaken in order to provide a framework on which to base the design of chemical compounds that inhibit the enzyme activity.A 693-bp fragment consistent with the size expected for the predicted cmk coding sequence was PCR amplified from M. tuberculosis H37Rv genomic DNA (see Fig. S1A in the supplemental material). For directional cloning, a forward primer (5Ј-GGC ATATGAGTCGCCTAAGCGCAGCGGTAGT-3Ј) and a reverse primer (5Ј-GTGGATCCTCACCGCACTGCCTCACTT CGC-3Ј) were designed to contain, respectively, NdeI and BamHI restriction sites (underlined). The PCR fragment was purified and ligated into the pET-23a(ϩ) expression vector (Novagen), and its sequence was determined by automated DNA sequencing. The resulting pET-23a(ϩ)::cmk plasmid was introduced into Escherichia coli BL21(DE3) by electroporation and selected on LB plates containing 50 g/ml ampicillin.

Kinetic mechanism and energetics of binding of phosphoryl group acceptors to Mycobacterium tuberculosis cytidine monophosphate kinase

“…Total protein concentration was determined by the method of Bradford [20], using the Bio-Rad protein assay kit (Bio-Rad Laboratories) and bovine serum albumin as standard. As described elsewhere [11], a typical yield of 5 mg of homogeneous recombinant MtCMK per liter of cell culture could be obtained.…”

“…Electrocompetent BL21(DE3) Escherichia coli cells were transformed with pET-23a(+)::cmk recombinant plasmid, and cell growth and purification steps of MtCMK were as described elsewhere [11]. The pooled fractions were dialyzed against 50 mM Tris-HCl pH 7.5, 50 mM KCl and 5 mM MgCl 2 , concentrated using an AMICON (Millipore Corporation, Bedford, MA) ultrafiltration membrane (MWCO = 10 kDa), and stored at À80°C.…”

“…We have previously reported that cmk gene in M. tuberculosis encodes a protein with CMK activity (MtCMK), which catalyzes the phosphoryl transfer from ATP to dCMP, CMP or uridine 5 0 -monophosphate (UMP) to form the corresponding nucleoside diphosphates [11]. However, MtCMK preferentially phosphorylates dCMP and CMP, whereas UMP is a poor substrate [11] (Fig. 1).…”

“…Bacterial cytidine 5 0 -monophosphate (CMP) kinase (CMK), which is part of pyrimidine nucleotide interconversion pathways, catalyzes the c-phosphoryl group transfer from, usually, adenosine-5 0 -triphosphate (ATP) to either CMP or 2 0 -deoxy-CMP (dCMP). We have previously reported that cmk gene in M. tuberculosis encodes a protein with CMK activity (MtCMK), which catalyzes the phosphoryl transfer from ATP to dCMP, CMP or uridine 5 0 -monophosphate (UMP) to form the corresponding nucleoside diphosphates [11]. However, MtCMK preferentially phosphorylates dCMP and CMP, whereas UMP is a poor substrate [11] (Fig.…”

“…In addition, CMK enzymes have been reported to be essential for different organisms: Streptococcus pneumonia [14], Bacillus subtilis [15], Salmonella typhimurium [16] and, probably, M. tuberculosis [7,8] due to its proposed role in recycling nucleotides derived from RNA degradation in the latter [11].…”