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Role of a cysteine residue in substrate entry and catalysis in MtHIBADH : Analysis by chemical modifications and site‐directed mutagenesis
Author(s) -
Singh Amrita,
Badepally Nagendar Goud,
Surolia Avadhesha
Publication year - 2021
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1002/iub.2466
Subject(s) - cysteine , active site , chemistry , biochemistry , dehydrogenase , enzyme , stereochemistry , alanine , cysteine metabolism , site directed mutagenesis , binding site , amino acid , mutant , gene
Despite sharing conserved substrate‐binding residues, members of 3‐hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol‐modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo‐first‐order kinetics as a function of the concentration of pCMB. S‐HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol‐modifying reagent. Site‐directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys‐210 residue, located at C‐terminal domain, for its dehydrogenase activity. Cys‐210 mutation to serine reduced the dehydrogenase activity by ~2‐fold while mutation to alanine strikingly reduced the activity by ~140‐fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys‐210 residue in inter‐chain interaction with Gln‐178, which acts as hydrogen bond donor and coordinates with Cys‐210 and Gly‐208 of the adjacent subunit. The data demonstrate a critical role of Cys‐210 residue in maintaining the conformation and rigidity of loop composed of substrate‐interacting residues involved in the entry of S‐HIBA substrate in MtHIBADH.