Solution structure and function of an essential CMP kinase of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity and has developed resistance to many antibiotics. We show that the gene product from SP1603, identified from S. pneumoniae TIGR4, is a CMP kinase that is essential for bacterial growth. It represents an attractive drug target for the development of a novel antibiotic to overcome the problems of drug resistance development for this organism. Here we describe the three-dimensional solution structure of the S. pneumoniae CMP kinase as determined by NMR spectroscopy. The structure consists of eight ␣-helices and two ␤-sheets that fold into the classical core domain, the substrate-binding domain, and the LID domain. The three domains of the protein pack together to form a central cavity for substrate-binding and enzymatic catalysis. The S. pneumoniae CMP kinase resembles the fold of the Escherichia coli homolog. An insertion of one residue is observed at the ␤-turn in the substrate-binding domain of the S. pneumoniae CMP kinase when compared with the E. coli homolog. Chemical shift perturbations caused by the binding of CMP, CDP, and ATP revealed that CMP or CDP binds to the junction between the core and substrate-binding domains, whereas ATP binds to the junction between the core and LID domains. From NMR relaxation studies, we determined that the loops in the LID domain are highly mobile. These mobile loops could aid in the closing/opening of the LID domain during enzyme catalysis.Keywords: Streptococcus pneumoniae CMP kinase; cytidylate kinase; cytidine monophosphate kinase; nucleoside monophosphate kinase; NMR structure; structural genomics Streptococcus pneumoniae is a major human pathogen that can cause potentially life-threatening community-acquired invasive diseases such as pneumonia, meningitis, and otitis media. S. pneumoniae has developed resistance to the antibiotics that are commonly used for its treatment (Schutze et al. 1994;Baquero 1995). One approach that is being used to address this problem is a full genome search for novel protein targets from this organism.From a genome-wide survey of the uncharacterized proteins from S. pneumoniae, we have identified several structural genomics targets. One of the targets is a protein corresponding to SP1603 in the recently determined complete genome sequence of a virulent isolate of S. pneumoniae (Tettelin et al. 2001) and to SPR1456 in the genome sequence of an avirulent strain of S. pneumoniae R6 (Hoskins et al. 2001). This protein has about 50% sequence homology with bacterial CMP kinases and thus could potentially be a CMP kinase. CMP kinase catalyzes the phosphoryl transfer from ATP to CMP and dCMP, resulting in the formation of nucleotide diphosphates. Therefore, CMP kinase plays a key role in cellular nucleic acid synthesis.Several crystal structures of CMP/UMP kinases have been determined including CMP kinase from Escherichia coli (Briozzo et al. 1998;Bertrand et al. 2002), UMP/CMP kinase from Dictyostelium discoideum (Scheffzek et al. 1996;Schlichting a...

Effect of 5-azacytidine on in vitro biofilm formation of Streptococcus pneumoniae

“…The cmK gene encodes cytidine kinase that catalyzes the phosphoryl transfer from ATP to cytidine monophosphokinase (CMP) and deoxycytidine monophosphokinase (dCMP), resulting in the formation of nucleotide diphosphates in cellular nucleic acid synthesis. A previous study found that cmK is an essential CMP kinase for bacterial growth and shares no sequence homology with the human ortholog [40]. The down regulated expression of cmK indicates that due the structural similarity of cytidine, 5-azacytidine interferes with DNA synthesis processes.…”

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

“…The slightly favorable entropic contribution is somewhat surprising as it has been reported that ATP binding induces conformational changes in the LID domain of CMKs [14,17]. It is thus tempting to suggest that the favorable overall entropic contribution is due to exclusion of water molecules from MtCMK active site, which should minimize the more favorable intramolecular hydrolysis of ATP to bound ADP and phosphate in the enzyme active site as compared to hydrolysis in solution [38].…”

“…6), and analysis of MtCMK structural model (Fig. 7) and of other bacterial CMK protein structures [13,14,42] suggest that the chemical groups that may contribute to the proton uptake effect include the 3-imino group of pyrimidine ring (pK a of 4.26), and/or the side chain(s) of Asp134 and Asp187 (Fig. 7) located in the NMP binding site (pK a value of 3.9-4.0 for the b-carboxyl group of aspartate in solution).…”

“…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].…”

Insight from the structural molecular model of cytidylate kinase from Mycobacterium tuberculosis

“…Bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) catalyses the phosphoryl transfer from ATP to CMP and dCMP, resulting in the formation of nucleoside diphosphates. CMP kinase has been shown to be necessary for bacterial growth in Streptococcus pneumoniae [10]. The Staphylococcus aureus CMP kinase crystal structure has been recently resolved [11].…”