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The structure and mechanism of the Mycobacterium tuberculosis cyclodityrosine synthetase.

Vetting MW, Hegde SS, Blanchard JS - Nat. Chem. Biol. (2010)

Bottom Line: The Mycobacterium tuberculosis enzyme Rv2275 catalyzes the formation of cyclo(L-Tyr-L-Tyr) using two molecules of Tyr-tRNA(Tyr) as substrates.The three-dimensional (3D) structure of Rv2275 was determined to 2.0-Å resolution, revealing that Rv2275 is structurally related to the class Ic aminoacyl-tRNA synthetase family of enzymes.Mutagenesis and radioactive labeling suggests a covalent intermediate in which L-tyrosine is transferred from Tyr-tRNA(Tyr) to an active site serine (Ser88) by transesterification with Glu233 serving as a critical base, catalyzing dipeptide bond formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.

ABSTRACT
The Mycobacterium tuberculosis enzyme Rv2275 catalyzes the formation of cyclo(L-Tyr-L-Tyr) using two molecules of Tyr-tRNA(Tyr) as substrates. The three-dimensional (3D) structure of Rv2275 was determined to 2.0-Å resolution, revealing that Rv2275 is structurally related to the class Ic aminoacyl-tRNA synthetase family of enzymes. Mutagenesis and radioactive labeling suggests a covalent intermediate in which L-tyrosine is transferred from Tyr-tRNA(Tyr) to an active site serine (Ser88) by transesterification with Glu233 serving as a critical base, catalyzing dipeptide bond formation.

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Related in: MedlinePlus

Cyclodityrosine synthetase reaction and 3-dimensional structure(a) Reaction scheme for synthesis of cyclodityrosine in Mycobacterium tuberculosis. Ribbon diagrams of (b) Rv2275, (c) catalytic domain of MjTyrRS. The terminal ends of a mobile loop not seen in the Rv2275 structure are labeled with red diamonds. The CP1 domain, a series of α helices located in the middle of the Rossmann fold of type I aa-tRNA synthetases, is colored green. (d) Stick diagram of residues around the two sequence motifs of CDPSs (H82x[LVI][LVI]G86[LVI]S88 and Y229[LVI]xxE233xP235) and which are proposed to be involved in binding and catalysis. Residues at the base, the rim and the periphery of the pocket are colored with cyan, yellow, and salmon carbons respectively. A dotted line is shown to illustrate the hydrogen bond between S88 and Y253. (e) Model of the S88-tyrosyl ester with the tyrosyl group bound to the internal pocket, and S88 in its observed position. Tyrosine ester shown with yellow carbons and S88 with green carbons. Potential hydrogen bonds between E233, Y229 and the tyrosyl amide shown as dashes. The side chain of N251 is approximately 4.5 Å from the tyrosyl hydroxyl and therefore the dashed lines are for illustrative purposes only.
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Figure 1: Cyclodityrosine synthetase reaction and 3-dimensional structure(a) Reaction scheme for synthesis of cyclodityrosine in Mycobacterium tuberculosis. Ribbon diagrams of (b) Rv2275, (c) catalytic domain of MjTyrRS. The terminal ends of a mobile loop not seen in the Rv2275 structure are labeled with red diamonds. The CP1 domain, a series of α helices located in the middle of the Rossmann fold of type I aa-tRNA synthetases, is colored green. (d) Stick diagram of residues around the two sequence motifs of CDPSs (H82x[LVI][LVI]G86[LVI]S88 and Y229[LVI]xxE233xP235) and which are proposed to be involved in binding and catalysis. Residues at the base, the rim and the periphery of the pocket are colored with cyan, yellow, and salmon carbons respectively. A dotted line is shown to illustrate the hydrogen bond between S88 and Y253. (e) Model of the S88-tyrosyl ester with the tyrosyl group bound to the internal pocket, and S88 in its observed position. Tyrosine ester shown with yellow carbons and S88 with green carbons. Potential hydrogen bonds between E233, Y229 and the tyrosyl amide shown as dashes. The side chain of N251 is approximately 4.5 Å from the tyrosyl hydroxyl and therefore the dashed lines are for illustrative purposes only.

Mentions: It was recently reported that the Streptomyces noursei protein AlbC catalyzes the formation of the albonoursin cyclodipeptide precursor cyclo(L-Phe-L-Leu) (cFL) using aminoacyl-tRNAs9. AlbC is a 239-residue polypeptide that is unrelated not only to NRPSs but also to all other structurally and functionally characterized proteins. In silico analysis of gene databases identified 7 other proteins exhibiting sequence similarity to AlbC. Mycobacterium tuberculosis has a chromosomally encoded AlbC homolog (Rv2275) exhibiting 26% sequence identity to AlbC. Recombinant M. tuberculosis Rv2275 catalyzed the Tyr-tRNA-dependent formation of cYY in E. coli cell lysates9. In M. tuberculosis, the adjacent gene encodes a cytochrome P450 (Rv2276) that catalyzes C-C bond formation between the carbons ortho to the phenolic hydroxyl of cYY, producing what we term mycocyclosin (Fig. 1a)10. Rv2276 was found to be an essential M. tuberculosis gene and it was suggested that either mycocyclosin was essential, or that the overproduction of cYY is toxic10,11.


The structure and mechanism of the Mycobacterium tuberculosis cyclodityrosine synthetase.

Vetting MW, Hegde SS, Blanchard JS - Nat. Chem. Biol. (2010)

Cyclodityrosine synthetase reaction and 3-dimensional structure(a) Reaction scheme for synthesis of cyclodityrosine in Mycobacterium tuberculosis. Ribbon diagrams of (b) Rv2275, (c) catalytic domain of MjTyrRS. The terminal ends of a mobile loop not seen in the Rv2275 structure are labeled with red diamonds. The CP1 domain, a series of α helices located in the middle of the Rossmann fold of type I aa-tRNA synthetases, is colored green. (d) Stick diagram of residues around the two sequence motifs of CDPSs (H82x[LVI][LVI]G86[LVI]S88 and Y229[LVI]xxE233xP235) and which are proposed to be involved in binding and catalysis. Residues at the base, the rim and the periphery of the pocket are colored with cyan, yellow, and salmon carbons respectively. A dotted line is shown to illustrate the hydrogen bond between S88 and Y253. (e) Model of the S88-tyrosyl ester with the tyrosyl group bound to the internal pocket, and S88 in its observed position. Tyrosine ester shown with yellow carbons and S88 with green carbons. Potential hydrogen bonds between E233, Y229 and the tyrosyl amide shown as dashes. The side chain of N251 is approximately 4.5 Å from the tyrosyl hydroxyl and therefore the dashed lines are for illustrative purposes only.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Cyclodityrosine synthetase reaction and 3-dimensional structure(a) Reaction scheme for synthesis of cyclodityrosine in Mycobacterium tuberculosis. Ribbon diagrams of (b) Rv2275, (c) catalytic domain of MjTyrRS. The terminal ends of a mobile loop not seen in the Rv2275 structure are labeled with red diamonds. The CP1 domain, a series of α helices located in the middle of the Rossmann fold of type I aa-tRNA synthetases, is colored green. (d) Stick diagram of residues around the two sequence motifs of CDPSs (H82x[LVI][LVI]G86[LVI]S88 and Y229[LVI]xxE233xP235) and which are proposed to be involved in binding and catalysis. Residues at the base, the rim and the periphery of the pocket are colored with cyan, yellow, and salmon carbons respectively. A dotted line is shown to illustrate the hydrogen bond between S88 and Y253. (e) Model of the S88-tyrosyl ester with the tyrosyl group bound to the internal pocket, and S88 in its observed position. Tyrosine ester shown with yellow carbons and S88 with green carbons. Potential hydrogen bonds between E233, Y229 and the tyrosyl amide shown as dashes. The side chain of N251 is approximately 4.5 Å from the tyrosyl hydroxyl and therefore the dashed lines are for illustrative purposes only.
Mentions: It was recently reported that the Streptomyces noursei protein AlbC catalyzes the formation of the albonoursin cyclodipeptide precursor cyclo(L-Phe-L-Leu) (cFL) using aminoacyl-tRNAs9. AlbC is a 239-residue polypeptide that is unrelated not only to NRPSs but also to all other structurally and functionally characterized proteins. In silico analysis of gene databases identified 7 other proteins exhibiting sequence similarity to AlbC. Mycobacterium tuberculosis has a chromosomally encoded AlbC homolog (Rv2275) exhibiting 26% sequence identity to AlbC. Recombinant M. tuberculosis Rv2275 catalyzed the Tyr-tRNA-dependent formation of cYY in E. coli cell lysates9. In M. tuberculosis, the adjacent gene encodes a cytochrome P450 (Rv2276) that catalyzes C-C bond formation between the carbons ortho to the phenolic hydroxyl of cYY, producing what we term mycocyclosin (Fig. 1a)10. Rv2276 was found to be an essential M. tuberculosis gene and it was suggested that either mycocyclosin was essential, or that the overproduction of cYY is toxic10,11.

Bottom Line: The Mycobacterium tuberculosis enzyme Rv2275 catalyzes the formation of cyclo(L-Tyr-L-Tyr) using two molecules of Tyr-tRNA(Tyr) as substrates.The three-dimensional (3D) structure of Rv2275 was determined to 2.0-Å resolution, revealing that Rv2275 is structurally related to the class Ic aminoacyl-tRNA synthetase family of enzymes.Mutagenesis and radioactive labeling suggests a covalent intermediate in which L-tyrosine is transferred from Tyr-tRNA(Tyr) to an active site serine (Ser88) by transesterification with Glu233 serving as a critical base, catalyzing dipeptide bond formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.

ABSTRACT
The Mycobacterium tuberculosis enzyme Rv2275 catalyzes the formation of cyclo(L-Tyr-L-Tyr) using two molecules of Tyr-tRNA(Tyr) as substrates. The three-dimensional (3D) structure of Rv2275 was determined to 2.0-Å resolution, revealing that Rv2275 is structurally related to the class Ic aminoacyl-tRNA synthetase family of enzymes. Mutagenesis and radioactive labeling suggests a covalent intermediate in which L-tyrosine is transferred from Tyr-tRNA(Tyr) to an active site serine (Ser88) by transesterification with Glu233 serving as a critical base, catalyzing dipeptide bond formation.

Show MeSH
Related in: MedlinePlus