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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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Proposed mechanism of cyclodipeptide formation for cyclodipeptide synthetasesShown is the mechanism where the dipeptide ester is formed on the tRNA. An equivalent argument could be made for the formation of the dipeptide ester of S88, however the roles of the enzyme groups would not change.
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Figure 3: Proposed mechanism of cyclodipeptide formation for cyclodipeptide synthetasesShown is the mechanism where the dipeptide ester is formed on the tRNA. An equivalent argument could be made for the formation of the dipeptide ester of S88, however the roles of the enzyme groups would not change.

Mentions: Based on the structure and our mutational analysis we propose catalysis occurs with the initial binding of Tyr-tRNATyr in an orientation such that α-amino group of tyrosine is positioned to allow for interaction with E233 (Fig. 3a). Nucleophilic attack by the S88 hydroxyl on the ester carbonyl results in the formation of the covalently tyrosinoylated enzyme and free Tyr-tRNA (Fig. 3b). In order for a second Tyr-tRNATyr to bind, the covalently bound tyrosine must swing out of its original binding pocket and the rotation of the side chain of S88 (from chi 1 = 55° to chi1 = 178°) would place the tyrosine in a large secondary surface depression (Supplementary Fig. 7). The formation of radiolabled tyrosinoylated enzyme with the E233Q mutant form of Rv2275 suggests that this initial transesterification chemistry does not require E233. The second Tyr-tRNATyr binds and the chemistry here becomes ambiguous. The α-amino group of the enzyme-bound tyrosine could attack the carbonyl ester of the Tyr-tRNATyr to generate the enzyme-bound dipeptide or the α-amino group of the tRNA-bound tyrosine could attack the enzyme bound tyrosine to generate the tRNA-bound dipeptide. The structure suggests that the latter is more likely, given the need for a general base to deprotonate the α-amino group to attack the enzyme ester bond. Once the first peptide bond is formed, the second chemical step occurs, and in this step E233 could assist in protonating the product 3′-hydroxyl group of tRNA. It is unclear how the protein plays a role in orienting the dipeptide to promote this cyclization chemistry, and although the intramolecular cyclization of a dipeptide ester is facile, it requires that the dipeptide be in a cis conformation to place the amine in proximity to the ester15,16.


The structure and mechanism of the Mycobacterium tuberculosis cyclodityrosine synthetase.

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

Proposed mechanism of cyclodipeptide formation for cyclodipeptide synthetasesShown is the mechanism where the dipeptide ester is formed on the tRNA. An equivalent argument could be made for the formation of the dipeptide ester of S88, however the roles of the enzyme groups would not change.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2957485&req=5

Figure 3: Proposed mechanism of cyclodipeptide formation for cyclodipeptide synthetasesShown is the mechanism where the dipeptide ester is formed on the tRNA. An equivalent argument could be made for the formation of the dipeptide ester of S88, however the roles of the enzyme groups would not change.
Mentions: Based on the structure and our mutational analysis we propose catalysis occurs with the initial binding of Tyr-tRNATyr in an orientation such that α-amino group of tyrosine is positioned to allow for interaction with E233 (Fig. 3a). Nucleophilic attack by the S88 hydroxyl on the ester carbonyl results in the formation of the covalently tyrosinoylated enzyme and free Tyr-tRNA (Fig. 3b). In order for a second Tyr-tRNATyr to bind, the covalently bound tyrosine must swing out of its original binding pocket and the rotation of the side chain of S88 (from chi 1 = 55° to chi1 = 178°) would place the tyrosine in a large secondary surface depression (Supplementary Fig. 7). The formation of radiolabled tyrosinoylated enzyme with the E233Q mutant form of Rv2275 suggests that this initial transesterification chemistry does not require E233. The second Tyr-tRNATyr binds and the chemistry here becomes ambiguous. The α-amino group of the enzyme-bound tyrosine could attack the carbonyl ester of the Tyr-tRNATyr to generate the enzyme-bound dipeptide or the α-amino group of the tRNA-bound tyrosine could attack the enzyme bound tyrosine to generate the tRNA-bound dipeptide. The structure suggests that the latter is more likely, given the need for a general base to deprotonate the α-amino group to attack the enzyme ester bond. Once the first peptide bond is formed, the second chemical step occurs, and in this step E233 could assist in protonating the product 3′-hydroxyl group of tRNA. It is unclear how the protein plays a role in orienting the dipeptide to promote this cyclization chemistry, and although the intramolecular cyclization of a dipeptide ester is facile, it requires that the dipeptide be in a cis conformation to place the amine in proximity to the ester15,16.

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