Purification and molecular characterization of a novel diadenosine 5',5'''-P(1),P(4)-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv.
In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5'-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5'-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K(m) values for Ap4A and phosphate were 0.10 and 0.94mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.
Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo 208-0011, Japan.
Shigetarou
S
Mori
Keigo
K
Shibayama
Jun-Ichi
J
Wachino
Yoshichika
Y
Arakawa
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D002413:Cations, Divalent; D008670:Metals; D009711:Nucleotides; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; C044740:diadenosine 5,5'''-P(1),P(4)-tetraphosphate alpha,beta-phosphorylase