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Structural and Mutational Studies on Substrate Specificity and Catalysis of Salmonella typhimurium D-Cysteine Desulfhydrase

Bharath, SakshibeeduR and Bisht, Shveta and Harijan, RajeshK and Savithri, HandanahalS and Murthy, MathurRN (2012) Structural and Mutational Studies on Substrate Specificity and Catalysis of Salmonella typhimurium D-Cysteine Desulfhydrase. In: PLOS ONE, 7 (5).

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Official URL: http://dx.doi.org/10.1371/journal.pone.0036267

Abstract

Salmonella typhimurium DCyD (StDCyD) is a fold type II pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. It also efficiently degrades beta-chloro-D-alanine (beta CDA). D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC). Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, beta CDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS) at resolutions ranging from 1.7 to 2.6 angstrom. The polypeptide fold of StDCyD consisting of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and beta CDA show the product, pyruvate, bound at a site 4.0-6.0 angstrom away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at C alpha proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for C alpha proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.

Item Type: Journal Article
Additional Information: Copy right for this article belongs to Public Library Science
Keywords: D-SERINE DEHYDRATASE;RING-OPENING REACTION; 1-AMINOCYCLOPROPANE-1-CARBOXYLATE DEAMINASE;3-DIMENSIONAL STRUCTURE;DIRECTED MUTAGENESIS;THREONINE DEAMINASE; SECONDARY-STRUCTURE;CRYSTAL-STRUCTURES;ENZYMES; IDENTIFICATION
Department/Centre: Division of Biological Sciences > Biochemistry
Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 17 Jul 2012 11:29
Last Modified: 17 Jul 2012 11:32
URI: http://eprints.iisc.ernet.in/id/eprint/44841

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