Toward the catalytic mechanism of a cysteine ligase (MshC) from Mycobacterium smegmatis

An enzyme involved in the biosynthetic pathway of mycothiol

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Abstract

Mycobacterium tuberculosis and other members of the actinomycete family produce mycothiol (MSH or acetylcysteine-glucosamine-inositol, AcCys-GlcN-Ins) to protect the organism against oxidative and antibiotic stress. The biosynthesis of MSH proceeds via a five-step process that involves four unique enzymes, MshA-D, which represent specific targets for inhibitor design. Recombinant Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of glucosamine-inositol (GlcN-Ins) and cysteine to form Cys-GlcN-Ins. The 1.6 Å three-dimensional structure of MshC in complex with a tight binding bisubstrate analogue, 5′-O-[N-(L-cysteinyl) sulfamonyl]adenosine (CSA), has suggested specific roles for T46, H55, T83, W227, and D251. In addition, a catalytic role for H55 has been proposed on the basis of studies of related aminoacyl-tRNA synthetases. Site-directed mutagenesis was conducted to evaluate the functional roles of these highly conserved residues. All mutants exhibited significantly decreased k cat values, with the exception of T83V for which a <7-fold decrease was observed compared to that of the wild type (WT). For the T46V, H55A, W227F, and D251N mutants, the rate of cysteine activation decreased 100-1400-fold compared to that of WT, consistent with the important roles of these residues in the first half-reaction. The ∼2000-fold decrease in k cat/Km as well as the ∼20-fold decrease in K m for cysteine suggested a significant role for T46 in cysteine binding. Kinetic studies also indicate a function for W227 in cysteine binding but not in substrate discrimination against serine. H55 was also observed to play a significant role in ATP binding as well as cysteine adenylation. The activity of H55A was partially rescued with exogenous imidazole at acidic pH values, suggesting that the protonated form of histidine is exerting a catalytic role. The pH dependence of the kinetic parameters with the WT enzyme suggests an additional requirement for a catalytic base in cysteinyl ligation.

Original languageEnglish (US)
Pages (from-to)7150-7159
Number of pages10
JournalBiochemistry
Volume48
Issue number30
DOIs
StatePublished - Aug 4 2009

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Mycobacterium smegmatis
Biosynthetic Pathways
Ligases
Cysteine
Glucosamine
Inositol
Enzymes
Melanocyte-Stimulating Hormones
4 alpha-glucanotransferase
Cats
Adenosine Triphosphate
Amino Acyl-tRNA Synthetases
Mutagenesis
Actinobacteria
Biosynthesis
Acetylcysteine
Site-Directed Mutagenesis
Mycobacterium tuberculosis
Kinetic parameters
Histidine

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{9371c4be6d3147dba84fff3e3607b1c0,
title = "Toward the catalytic mechanism of a cysteine ligase (MshC) from Mycobacterium smegmatis: An enzyme involved in the biosynthetic pathway of mycothiol",
abstract = "Mycobacterium tuberculosis and other members of the actinomycete family produce mycothiol (MSH or acetylcysteine-glucosamine-inositol, AcCys-GlcN-Ins) to protect the organism against oxidative and antibiotic stress. The biosynthesis of MSH proceeds via a five-step process that involves four unique enzymes, MshA-D, which represent specific targets for inhibitor design. Recombinant Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of glucosamine-inositol (GlcN-Ins) and cysteine to form Cys-GlcN-Ins. The 1.6 {\AA} three-dimensional structure of MshC in complex with a tight binding bisubstrate analogue, 5′-O-[N-(L-cysteinyl) sulfamonyl]adenosine (CSA), has suggested specific roles for T46, H55, T83, W227, and D251. In addition, a catalytic role for H55 has been proposed on the basis of studies of related aminoacyl-tRNA synthetases. Site-directed mutagenesis was conducted to evaluate the functional roles of these highly conserved residues. All mutants exhibited significantly decreased k cat values, with the exception of T83V for which a <7-fold decrease was observed compared to that of the wild type (WT). For the T46V, H55A, W227F, and D251N mutants, the rate of cysteine activation decreased 100-1400-fold compared to that of WT, consistent with the important roles of these residues in the first half-reaction. The ∼2000-fold decrease in k cat/Km as well as the ∼20-fold decrease in K m for cysteine suggested a significant role for T46 in cysteine binding. Kinetic studies also indicate a function for W227 in cysteine binding but not in substrate discrimination against serine. H55 was also observed to play a significant role in ATP binding as well as cysteine adenylation. The activity of H55A was partially rescued with exogenous imidazole at acidic pH values, suggesting that the protonated form of histidine is exerting a catalytic role. The pH dependence of the kinetic parameters with the WT enzyme suggests an additional requirement for a catalytic base in cysteinyl ligation.",
author = "Fan Fan and Blanchard, {John S.}",
year = "2009",
month = "8",
day = "4",
doi = "10.1021/bi900457x",
language = "English (US)",
volume = "48",
pages = "7150--7159",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "30",

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T1 - Toward the catalytic mechanism of a cysteine ligase (MshC) from Mycobacterium smegmatis

T2 - An enzyme involved in the biosynthetic pathway of mycothiol

AU - Fan, Fan

AU - Blanchard, John S.

PY - 2009/8/4

Y1 - 2009/8/4

N2 - Mycobacterium tuberculosis and other members of the actinomycete family produce mycothiol (MSH or acetylcysteine-glucosamine-inositol, AcCys-GlcN-Ins) to protect the organism against oxidative and antibiotic stress. The biosynthesis of MSH proceeds via a five-step process that involves four unique enzymes, MshA-D, which represent specific targets for inhibitor design. Recombinant Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of glucosamine-inositol (GlcN-Ins) and cysteine to form Cys-GlcN-Ins. The 1.6 Å three-dimensional structure of MshC in complex with a tight binding bisubstrate analogue, 5′-O-[N-(L-cysteinyl) sulfamonyl]adenosine (CSA), has suggested specific roles for T46, H55, T83, W227, and D251. In addition, a catalytic role for H55 has been proposed on the basis of studies of related aminoacyl-tRNA synthetases. Site-directed mutagenesis was conducted to evaluate the functional roles of these highly conserved residues. All mutants exhibited significantly decreased k cat values, with the exception of T83V for which a <7-fold decrease was observed compared to that of the wild type (WT). For the T46V, H55A, W227F, and D251N mutants, the rate of cysteine activation decreased 100-1400-fold compared to that of WT, consistent with the important roles of these residues in the first half-reaction. The ∼2000-fold decrease in k cat/Km as well as the ∼20-fold decrease in K m for cysteine suggested a significant role for T46 in cysteine binding. Kinetic studies also indicate a function for W227 in cysteine binding but not in substrate discrimination against serine. H55 was also observed to play a significant role in ATP binding as well as cysteine adenylation. The activity of H55A was partially rescued with exogenous imidazole at acidic pH values, suggesting that the protonated form of histidine is exerting a catalytic role. The pH dependence of the kinetic parameters with the WT enzyme suggests an additional requirement for a catalytic base in cysteinyl ligation.

AB - Mycobacterium tuberculosis and other members of the actinomycete family produce mycothiol (MSH or acetylcysteine-glucosamine-inositol, AcCys-GlcN-Ins) to protect the organism against oxidative and antibiotic stress. The biosynthesis of MSH proceeds via a five-step process that involves four unique enzymes, MshA-D, which represent specific targets for inhibitor design. Recombinant Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of glucosamine-inositol (GlcN-Ins) and cysteine to form Cys-GlcN-Ins. The 1.6 Å three-dimensional structure of MshC in complex with a tight binding bisubstrate analogue, 5′-O-[N-(L-cysteinyl) sulfamonyl]adenosine (CSA), has suggested specific roles for T46, H55, T83, W227, and D251. In addition, a catalytic role for H55 has been proposed on the basis of studies of related aminoacyl-tRNA synthetases. Site-directed mutagenesis was conducted to evaluate the functional roles of these highly conserved residues. All mutants exhibited significantly decreased k cat values, with the exception of T83V for which a <7-fold decrease was observed compared to that of the wild type (WT). For the T46V, H55A, W227F, and D251N mutants, the rate of cysteine activation decreased 100-1400-fold compared to that of WT, consistent with the important roles of these residues in the first half-reaction. The ∼2000-fold decrease in k cat/Km as well as the ∼20-fold decrease in K m for cysteine suggested a significant role for T46 in cysteine binding. Kinetic studies also indicate a function for W227 in cysteine binding but not in substrate discrimination against serine. H55 was also observed to play a significant role in ATP binding as well as cysteine adenylation. The activity of H55A was partially rescued with exogenous imidazole at acidic pH values, suggesting that the protonated form of histidine is exerting a catalytic role. The pH dependence of the kinetic parameters with the WT enzyme suggests an additional requirement for a catalytic base in cysteinyl ligation.

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