Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium

Comparison of A and B isozymes

Arundhati Chattopadhyay, Markus Meier, Sergei Ivaninskii, Peter Burkhard, Francesca Speroni, Barbara Campanini, Stefano Bettati, Andrea Mozzarelli, Wael Rabeh, Lei Li, Paul F. Cook

    Research output: Contribution to journalArticle

    Abstract

    O-Acetylserine sulfhydrylase is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the β-acetoxy group of O-acetyl-L-serine by a thiol to give L-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 Å and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 Å from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an ∼10-fold lower Km for O-acetyl-L-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the α-aminoacrylate intermediate at any concentration of O-acetyl-L-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the α-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the α-amine's pKa. The pH dependence of the first-order rate constant for decay of the α-aminoacrylate intermediate to give pyruvate and ammonia gives a pK a of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with L-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the α-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.

    Original languageEnglish (US)
    Pages (from-to)8315-8330
    Number of pages16
    JournalBiochemistry
    Volume46
    Issue number28
    DOIs
    StatePublished - Jul 17 2007

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    Cysteine Synthase
    Salmonella
    Salmonella typhimurium
    Isoenzymes
    Serine
    Cysteine
    Schiff Bases
    Catalytic Domain
    Titration
    Amines
    Acetates
    Enzymes
    Pyridoxal Phosphate
    Biosynthetic Pathways
    Enterobacteriaceae
    Hydrogen Bonding

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Chattopadhyay, A., Meier, M., Ivaninskii, S., Burkhard, P., Speroni, F., Campanini, B., ... Cook, P. F. (2007). Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: Comparison of A and B isozymes. Biochemistry, 46(28), 8315-8330. https://doi.org/10.1021/bi602603c

    Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium : Comparison of A and B isozymes. / Chattopadhyay, Arundhati; Meier, Markus; Ivaninskii, Sergei; Burkhard, Peter; Speroni, Francesca; Campanini, Barbara; Bettati, Stefano; Mozzarelli, Andrea; Rabeh, Wael; Li, Lei; Cook, Paul F.

    In: Biochemistry, Vol. 46, No. 28, 17.07.2007, p. 8315-8330.

    Research output: Contribution to journalArticle

    Chattopadhyay, A, Meier, M, Ivaninskii, S, Burkhard, P, Speroni, F, Campanini, B, Bettati, S, Mozzarelli, A, Rabeh, W, Li, L & Cook, PF 2007, 'Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: Comparison of A and B isozymes', Biochemistry, vol. 46, no. 28, pp. 8315-8330. https://doi.org/10.1021/bi602603c
    Chattopadhyay, Arundhati ; Meier, Markus ; Ivaninskii, Sergei ; Burkhard, Peter ; Speroni, Francesca ; Campanini, Barbara ; Bettati, Stefano ; Mozzarelli, Andrea ; Rabeh, Wael ; Li, Lei ; Cook, Paul F. / Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium : Comparison of A and B isozymes. In: Biochemistry. 2007 ; Vol. 46, No. 28. pp. 8315-8330.
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    title = "Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: Comparison of A and B isozymes",
    abstract = "O-Acetylserine sulfhydrylase is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the β-acetoxy group of O-acetyl-L-serine by a thiol to give L-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 {\AA} and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 {\AA} from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an ∼10-fold lower Km for O-acetyl-L-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the α-aminoacrylate intermediate at any concentration of O-acetyl-L-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the α-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the α-amine's pKa. The pH dependence of the first-order rate constant for decay of the α-aminoacrylate intermediate to give pyruvate and ammonia gives a pK a of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with L-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the α-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.",
    author = "Arundhati Chattopadhyay and Markus Meier and Sergei Ivaninskii and Peter Burkhard and Francesca Speroni and Barbara Campanini and Stefano Bettati and Andrea Mozzarelli and Wael Rabeh and Lei Li and Cook, {Paul F.}",
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    TY - JOUR

    T1 - Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium

    T2 - Comparison of A and B isozymes

    AU - Chattopadhyay, Arundhati

    AU - Meier, Markus

    AU - Ivaninskii, Sergei

    AU - Burkhard, Peter

    AU - Speroni, Francesca

    AU - Campanini, Barbara

    AU - Bettati, Stefano

    AU - Mozzarelli, Andrea

    AU - Rabeh, Wael

    AU - Li, Lei

    AU - Cook, Paul F.

    PY - 2007/7/17

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    N2 - O-Acetylserine sulfhydrylase is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the β-acetoxy group of O-acetyl-L-serine by a thiol to give L-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 Å and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 Å from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an ∼10-fold lower Km for O-acetyl-L-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the α-aminoacrylate intermediate at any concentration of O-acetyl-L-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the α-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the α-amine's pKa. The pH dependence of the first-order rate constant for decay of the α-aminoacrylate intermediate to give pyruvate and ammonia gives a pK a of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with L-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the α-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.

    AB - O-Acetylserine sulfhydrylase is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the β-acetoxy group of O-acetyl-L-serine by a thiol to give L-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 Å and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 Å from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an ∼10-fold lower Km for O-acetyl-L-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the α-aminoacrylate intermediate at any concentration of O-acetyl-L-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the α-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the α-amine's pKa. The pH dependence of the first-order rate constant for decay of the α-aminoacrylate intermediate to give pyruvate and ammonia gives a pK a of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with L-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the α-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.

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