The 1.6 Å crystal structure of Mycobacterium smegmatis MshC: The penultimate enzyme in the mycothiol biosynthetic pathway

L. W. Tremblay, F. Fan, M. W. Vetting, John S. Blanchard

Research output: Contribution to journalArticle

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Abstract

Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and L-cysteine to form L-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5′-O-[N-(L-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 Å. The refined structure exhibited electron density corresponding to the entire 47 kDa MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions.

Original languageEnglish (US)
Pages (from-to)13326-13335
Number of pages10
JournalBiochemistry
Volume47
Issue number50
DOIs
StatePublished - Dec 16 2008

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cysteinyl-tRNA synthetase
Mycobacterium smegmatis
Biosynthetic Pathways
Adenosine
Catalytic Domain
Crystal structure
Enzymes
Proteolysis
Biosynthesis
Substrates
Transfer RNA
Trypsin
Catalysis
Carrier concentration
Metal ions
Cysteine
Zinc
Condensation
Ions
Conservation

ASJC Scopus subject areas

  • Biochemistry

Cite this

The 1.6 Å crystal structure of Mycobacterium smegmatis MshC : The penultimate enzyme in the mycothiol biosynthetic pathway. / Tremblay, L. W.; Fan, F.; Vetting, M. W.; Blanchard, John S.

In: Biochemistry, Vol. 47, No. 50, 16.12.2008, p. 13326-13335.

Research output: Contribution to journalArticle

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abstract = "Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and L-cysteine to form L-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5′-O-[N-(L-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 {\AA}. The refined structure exhibited electron density corresponding to the entire 47 kDa MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions.",
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