Arginine biosynthesis pathway is crucial to the survival and pathogenesis of Mycobacterium tuberculosis (Mtb). Arginine is a critical amino acid that contributes to the inflection of cellular immune responses during pathogenesis. Argininosuccinate lyase from Mtb (MtArgH), the last enzyme in the pathway, catalyzes the production of arginine from argininosuccinic acid. MtArgH is an essential enzyme for the growth and survival of M. tuberculosis. We biochemically characterized MtArgH and deciphered the role of a previously unexplored cysteine (Cys441 ) residue at the C-terminal region of the protein. Chemical modification of Cys441 completely abrogated the enzymatic activity suggesting its involvement in the catalytic mechanism. Replacement of Cys441 to alanine showed a striking decrease in the enzymatic activity, while retaining the overall secondary to quaternary structure of the protein, hence corroborating the involvement of Cys441 in the process of catalysis. Interestingly, replacement of Cys441 to serine, showed significant increase in activity, as compared to the wild-type MtArgH. Inactivity of C441 A and elevated activity of its conservative mutant (C441 S) confirmed the participation of Cys441 in the MtArgH activity. We also, observed that C441 S mutant has higher thermal stability and maintains significant activity at high temperatures. This is in concordance with our observation that Cys441 in Mtb is replaced by a serine in the ArgH from thermophilic microorganisms. Furthermore, we also propose a potential feedback mechanism, wherein the Cys441 is covalently modified to S-(2-succinyl) cysteine (succination) by one of the products, fumarate, thereby inactivating MtArgH. These insights into the mechanism of MtArgH activity unravel novel regulations of arginine biosynthetic pathway in Mtb. © 2017 IUBMB Life, 69(11):896-907, 2017.
Keywords: Mycobacterium tuberculosis; arginine biosynthesis; argininosuccinate lyase; cysteine modification; enzyme catalysis.
© 2017 International Union of Biochemistry and Molecular Biology.