Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis

Singh, A and Badepally, NG and Surolia, A (2021) Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis. In: IUBMB Life .

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Abstract

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 à 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. © 2021 International Union of Biochemistry and Molecular Biology

Item Type:	Journal Article
Publication:	IUBMB Life
Publisher:	Blackwell Publishing Ltd
Additional Information:	The copyright for this article belongs to Blackwell Publishing Ltd
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	04 Jul 2021 05:44
Last Modified:	04 Jul 2021 05:44
URI:	http://eprints.iisc.ac.in/id/eprint/68739

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