A Change in the Internal Aldimine Lysine (K42) in O -Acetylserine Sulfhydrylase to Alanine Indicates Its Importance in Transimination and as a General Base Catalyst †

Duke University, Durham, North Carolina, United States
Biochemistry (Impact Factor: 3.02). 11/1996; 35(41):13485-93. DOI: 10.1021/bi961517j
Source: PubMed


O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes a beta-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide. The pyridoxal 5'-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine. In the present study, the Schiff base lysine was identified as lysine 42, and its role in the OASS reaction was determined by changing it to alanine using site-directed mutagenesis. K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates. Apo-K42A-OASS can be reconstituted with PLP, suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base. The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the alpha-aminoacrylate intermediate on addition of OAS, suggesting that K42 is involved in the abstraction of the alpha-proton in the beta-elimination reaction. The external aldimines formed upon addition of L-Ala or L-Ser are stable and represent a tautomer that absorbs maximally at 420 nm, while L-Cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, and is also seen in the overall reaction after addition of primary amines to the assay system. The use of a small primary amine such as ethylamine or bromoethylamine in the assay system leads to the initial formation of an internal (gamma-thialysine) or external (ethylamine) aldimine followed by the slow formation of the alpha-aminoacrylate intermediate on addition of OAS. Activity could not be fully recovered, and only a single turnover is observed. Data suggest a significant rate enhancement resulting from the presence of K42 for transimination and general base catalysis.

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Available from: Klaus Dieter Schnackerz, May 14, 2014
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    • "OASS follows a ping pong kinetic mechanism where the conserved catalytic lysine residue forms an internal aldimine with PLP in the native state. OAS substitutes for lysine at the active site and forms an external Schiff base with PLP, followed by β elimination in which acetate is released and a proton is abstracted from the α position [7]. This leads to the formation of the α amino acrylate intermediate covalently linked to PLP (Fig. 1). "
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    ABSTRACT: BACKGROUND: O-acetyl serine sulfhydrylase (OASS) is a pyridoxal phosphate (PLP) dependent enzyme catalyzing the last step of the cysteine biosynthetic pathway. Here we analyze and investigate the factors responsible for recognition and different conformational changes accompanying the binding of various ligands to OASS. METHODS: X ray crystallography was used to determine the structures of OASS from Entamoeba histolytica in complex with methionine (substrate analogue), isoleucine (inhibitor) and an inhibitory tetra peptide to 2.00 Å, 2.03 Å and 1.87 Å resolutions, respectively. Molecular dynamics simulations were used to investigate the reasons responsible for the extent of domain movement and cleft closure of the enzyme in presence of different ligands. RESULTS: Here we report for the first time an OASS-methionine structure with an unmutated catalytic lysine at the active site. This is also the first OASS structure with a closed active site lacking external aldimine formation. The OASS-isoleucine structure shows the active site cleft in open state. Molecular dynamics studies indicate that cofactor PLP, N88 and G192 form a triad of energy contributors to close the active site upon ligand binding and orientation of the Schiff base forming nitrogen of the ligand is critical for this interaction. CONCLUSIONS: Methionine proves to be a better binder to OASS than isoleucine. The β branching of isoleucine does not allow it to reorient itself in suitable conformation near PLP to cause active site closure. GENERAL SIGNIFICANCE: Our findings have important implications in designing better inhibitors against OASS across all pathogenic microbial species.
    Full-text · Article · Jun 2013 · Biochimica et Biophysica Acta
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    • "Binding of cysteine or methionine to OASS changes both tryptophan and PLP fluorescence spectra (Figure 1A &1B). Ligand binding quenches tryptophan fluorescence observed at 345 nm (Figure 1A), but increases the PLP fluorescence at 507 nm as observed earlier [26]. It was observed that excitation of OASS at 290 nm leads to fluorescence at 345 nm as well as at 500 nm due to energy transfer from tryptophan to PLP. "
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    ABSTRACT: The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine). Ligand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (ΔH(St-Met) = -1.5 ± 0.1 kJ/mol; TΔS(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity. We show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.
    Full-text · Article · Jun 2011 · BMC Biochemistry
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    ABSTRACT: The last step in cysteine biosynthesis in enteric bacteria is catalyzed by the pyridoxal 5′-phosphate-dependent enzyme O-acetylserine sulfhydrylase. Here we report the crystal structure at 2.2 Å resolution of the A-isozyme of O-acetylserine sulfhydrylase isolated from Salmonella typhimurium. O-acetylserine sulfhydrylase shares the same fold with tryptophan synthase-β from Salmonella typhimurium but the sequence identity level is below 20%. There are some major structural differences: the loops providing the interface to the α-subunit in tryptophan synthase-β and two surface helices of tryptophan synthase-β are missing in O-acetylserine sulfhydrylase. The hydrophobic channel for indole transport from the α to the β active site of tryptophan synthase-β is, not unexpectedly, also absent in O-acetylserine sulfhydrylase. The dimer interface, on the other hand, is more or less conserved in the two enzymes. The active site cleft of O-acetylserine sulfhydrylase is wider and therefore more exposed to the solvent. A possible binding site for the substrate O-acetylserine is discussed.
    Full-text · Article · Feb 1998 · Journal of Molecular Biology
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