Journal
BIOCHEMISTRY
Volume 47, Issue 21, Pages 5823-5831Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi702453s
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Funding
- NCRR NIH HHS [1S10RR022393-01] Funding Source: Medline
- NIAID NIH HHS [AI51622, R01 AI075637] Funding Source: Medline
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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 MgCl2. 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 mu M) 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.
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