Purification and characterization of a functionally active Mycobacterium tuberculosis prephenate dehydrogenase.
Tuberculosis (TB) remains to be a global health problem. New drugs are badly needed to drastically reduce treatment time and overcome some of the challenges with tuberculosis treatment, such as multi-drug resistant (MDR) strain infected patients or tuberculosis/HIV co-infected patients. The essentiality of mycobacterial aromatic amino acid biosynthesis pathways and their absence from human host indicate that the member enzymes of these pathways promising drug targets for therapeutic agents against pathogen mycobacteria. Prephenate dehydrogenase (PDH) is a key regulatory enzyme in tyrosine biosynthesis, catalyzing the NAD(+)-dependent conversion of prephenate to p-hydroxyphenylpyruvate, making it a potential drug target for antibiotics discovery. The recombinant PDH with an N-terminal His-tag (His-rMtPDH) was first purified in Escherichia coli, and using enterokinase rMtPDH was obtained by cleaving the N-terminal fusion partner. The effect of pH, temperature and the cation-Na(+) on purified enzyme activity was characterized. The N-terminal fusion partner was found to have little effect on the biochemical properties of PDH. We also provide in vitro evidence that Mycobacterium tuberculosis PDH does not possess any chorismate mutase (CM) activity, which suggests that, unlike many other enteric bacteria (where PDH exists as a fusion protein with CM), M. tuberculosis PDH is a monofunctional protein.
Xu
Shengfeng
S
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PR China.
Yang
Yanping
Y
Jin
Ruiliang
R
Zhang
Min
M
Wang
Honghai
H
D000890:Anti-Infective Agents / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015195:Drug Design; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000737:chemistry; D015658:HIV Infections / Q000150:complications / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D006801:Humans; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D009164:Mycobacterium Infections / Q000150:complications / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D011303:Prephenate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000096:biosynthesis / Q000235:genetics
D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C076398:TyrA protein, Bacteria; D014443:Tyrosine; D011303:Prephenate Dehydrogenase