Rv2131c from Mycobacterium tuberculosis is a CysQ 3'-phosphoadenosine-5'-phosphatase.
Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3'-phosphoadenosine-5'-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the Mtb genome suggests the presence of a CysQ homologue encoded by the gene Rv2131c. However, a recent biochemical study assigned the protein's function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed Rv2131c heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl 2. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a K m (8.1 +/- 3.1 microM) and k cat (5.4 +/- 1.1 s (-1)) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the Rv2131c-encoded enzyme to dephosphorylate PAP and PAPS in vivo was confirmed by functional complementation of an Escherichia coli Delta cysQ mutant. Taken together, these studies indicate that Rv2131c encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism.
Hatzios
Stavroula K
SK
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Iavarone
Anthony T
AT
Bertozzi
Carolyn R
CR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009708:Nucleotidases / Q000737:chemistry* / Q000502:physiology; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D001426:Bacterial Proteins; D010724:Phosphoadenosine Phosphosulfate; C510507:CysQ protein, E coli; D009708:Nucleotidases; D010744:Phosphoric Monoester Hydrolases; C053354:bisphosphoadenylate 3'-nucleotidase