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 β-chloro-D-alanine (βCDA). 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, βCDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS) at resolutions ranging from 1.7 to 2.6 Å. 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 βCDA show the product, pyruvate, bound at a site 4.0–6.0 Å 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 Cα 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 Cα proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.
Highlights
Pyridoxal 59 phosphate (PLP)-dependent enzymes catalyze several reactions in the metabolism of amino acids
The structures of Salmonella typhimurium D-cysteine desulfhydrase (DCyD) (StDCyD) and its complexes presented in this manuscript have provided significant insights on enzyme specificity and differences from other closely related fold type II PLPdependent enzymes such as ACCDs
StDCyD is specific for D-Cys and bCDA, and shows no activity with amino-1-carboxy cyclopropane (ACC), it shares significant sequence identity with ACCDs
Summary
Pyridoxal 59 phosphate (PLP)-dependent enzymes catalyze several reactions in the metabolism of amino acids. PLPdependent enzymes have been classified into three groups (a, b, and c families) based on the carbon atom at which the net reaction takes place [2]. They have been classified into four structurally distinct folds (I–IV) [3]. The majority of PLP-dependent enzymes that catalyze elimination and replacement reactions with amino acids are specific to L-amino acids. D-cysteine (D-Cys) moieties are found in firefly luciferin, semisynthetic cephalosporin MT141 and malformin A [5,6]. D-cysteine desulfhydrase (DCyD) is suggested to take part in the catabolic degradation of D-Cys and D-Cys-containing toxic compounds [7]
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