Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4- cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli

Ming Jiang, Xiaolei Chen, Zufeng Guo, Yang Cao, Minjiao Chen, Zhihong Guo

Research output: Contribution to journalArticle

Abstract

Menaquinone is a lipid-soluble molecule that plays an essential role as an electron carrier in the respiratory chain of many bacteria. We have previously shown that its biosynthesis in Escherichia coli involves a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and requires an additional enzyme to convert this intermediate into (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). Here, we report the identification and characterization of MenH (or YfbB), an enzyme previously proposed to catalyze a late step in menaquinone biosynthesis, as the SHCHC synthase. The synthase catalyzes a proton abstraction reaction that results in 2,5-elimination of pyruvate from SEPHCHC and the formation of SHCHC. It is an efficient enzyme (kcat/KM = 2.0 × 10 7 M-1 s-1) that provides a smaller transition-state stabilization than other enzymes catalyzing proton abstraction from carbon acids. Despite its lack of the proposed thioesterase activity, the SHCHC synthase is homologous to the well-characterized C-C bond hydrolase MhpC. The crystallographic structure of the Vibrio cholerae MenH protein closely resembles that of MhpC and contains a Ser-His-Asp triad typical of serine proteases. Interestingly, this triad is conserved in all MenH proteins and is essential for the SHCHC synthase activity. Mutational analysis found that the catalytic efficiency of the E. coli protein is reduced by 1.4 × 10 3, 2.1 × 105, and 9.3 × 103 folds when alanine replaces serine, histidine, and aspartate of the triad, respectively. These results show that the SHCHC synthase is closely related to α/β hydrolases but catalyzes a reaction mechanistically distinct from all known hydrolase reactions.

Original languageEnglish (US)
Pages (from-to)3426-3434
Number of pages9
JournalBiochemistry
Volume47
Issue number11
DOIs
StatePublished - Mar 18 2008
Externally publishedYes

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Vitamin K 2
Biosynthesis
Escherichia coli
Hydrolases
Enzymes
Protons
Vibrio cholerae
Escherichia coli Proteins
Serine Proteases
Electron Transport
Pyruvic Acid
Histidine
Aspartic Acid
Alanine
Serine
2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase
1,4-cyclohexadiene
Bacteria
Proteins
Carbon

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4- cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli. / Jiang, Ming; Chen, Xiaolei; Guo, Zufeng; Cao, Yang; Chen, Minjiao; Guo, Zhihong.

In: Biochemistry, Vol. 47, No. 11, 18.03.2008, p. 3426-3434.

Research output: Contribution to journalArticle

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abstract = "Menaquinone is a lipid-soluble molecule that plays an essential role as an electron carrier in the respiratory chain of many bacteria. We have previously shown that its biosynthesis in Escherichia coli involves a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and requires an additional enzyme to convert this intermediate into (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). Here, we report the identification and characterization of MenH (or YfbB), an enzyme previously proposed to catalyze a late step in menaquinone biosynthesis, as the SHCHC synthase. The synthase catalyzes a proton abstraction reaction that results in 2,5-elimination of pyruvate from SEPHCHC and the formation of SHCHC. It is an efficient enzyme (kcat/KM = 2.0 × 10 7 M-1 s-1) that provides a smaller transition-state stabilization than other enzymes catalyzing proton abstraction from carbon acids. Despite its lack of the proposed thioesterase activity, the SHCHC synthase is homologous to the well-characterized C-C bond hydrolase MhpC. The crystallographic structure of the Vibrio cholerae MenH protein closely resembles that of MhpC and contains a Ser-His-Asp triad typical of serine proteases. Interestingly, this triad is conserved in all MenH proteins and is essential for the SHCHC synthase activity. Mutational analysis found that the catalytic efficiency of the E. coli protein is reduced by 1.4 × 10 3, 2.1 × 105, and 9.3 × 103 folds when alanine replaces serine, histidine, and aspartate of the triad, respectively. These results show that the SHCHC synthase is closely related to α/β hydrolases but catalyzes a reaction mechanistically distinct from all known hydrolase reactions.",
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AU - Chen, Minjiao

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