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
ABSTRACT 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.
Full-textDOI: · Available from: Klaus Dieter Schnackerz, May 14, 2014
- SourceAvailable from: Samudrala Gourinath
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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 . This leads to the formation of the α amino acrylate intermediate covalently linked to PLP (Fig. 1). "
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.Biochimica et Biophysica Acta 06/2013; 1830(10). DOI:10.1016/j.bbagen.2013.05.041 · 4.66 Impact Factor
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ABSTRACT: The sulfur amino acids cysteine and methionine function in many basic and essential processes of life. For cysteine this includes structural, catalytic, regulatory and metabolic functions. The special redox chemistry of sulfur and the thiol group in particular proved to be a versatile tool during evolution, not the least in electron transport processes in association with iron. Plants are primary producers and carry out assimilatory sulfate reduction to first synthesize cysteine that subsequently forms the backbone for methionine formation. This reaction sequence seems to be conserved in all phototrophic organisms. The position of cysteine biosynthesis between assimilation of inorganic sulfate and metabolization of organic sulfide makes it a prime target for coordination of both complex processes. It is thus a mediator between supply and demand in sulfur metabolism of a cell. Much attention has been paid to cysteine biosynthesis in plants, while less is known about the pathway in algae, cyanobacteria and purple bacteria. Recent evidence indicates that the two enzymes of cysteine synthesis, serine acetyltransferase and O-acetylserine-(thiol)-lyase are highly conserved between these groups and, at least in plants, form a reversible protein complex. This so-called cysteine synthase complex has been suggested to act as sensor for sulfide in cells and to be part of a regulatory loop that maintains cysteine homeostasis between sulfate reduction and cysteine consumption. Kinetic studies of the properties of the enzymes together with structural modelling of the proteins in the cysteine synthase complex as well studies using transgenic plants strongly support this unique regulatory system in plants. The degradation of cysteine is still an under-investigated subject. Possible alternative routes of thiol transfer and sulfide release are compiled here and discussed with respect to their putative functions in S-transfer reactions, cysteine degradation, detoxification reactions and iron-sulfur cluster biosynthesis.pages 61-94; Springer.
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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.Journal of Molecular Biology 02/1998; 283(1-283):121-133. DOI:10.1006/jmbi.1998.2037 · 4.33 Impact Factor