Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis.
5'-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 2.4.2.28) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in Mycobacterium tuberculosis, was expressed in and purified from Escherichia coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-l-homocysteine were poor substrates and no activity was detected with 5'-methylthioinosine, the other natural purines, or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the K(m) value for MTA was 9 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicated that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure-activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, AL 35294, USA.
Kajal
K
Buckoreelall
Yanjie
Y
Sun
Judith V
JV
Hobrath
Landon
L
Wilson
William B
WB
Parker
D002384:Catalysis; D003839:Deoxyadenosines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D010802:Phylogeny; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D011687:Purines / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D013872:Thionucleosides / Q000494:pharmacology
D003839:Deoxyadenosines; D010710:Phosphates; D011687:Purines; D013872:Thionucleosides; C008500:5'-methylthioadenosine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase