Kinetic Analysis of the 4-Methylideneimidazole-5-one-Containing Tyrosine Aminomutase in Enediyne Antitumor Antibiotic C-1027 Biosynthesis

Steven D. Christenson, Weiming Wu, M. Ashley Spies, Ben Shen, Michael D. Toney

Research output: Contribution to journalArticle

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Abstract

The enediyne antitumor antibiotic C-1027 contains an unusual (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety, which requires an aminomutase for its biosynthesis. Previously, we established that SgcC4 is an aminomutase that catalyzes the conversion of L-tyrosine to (S)-β-tyrosine and employs 4-methylideneimidazole-5-one (MIO) at its active site [Christenson, S. D., Liu, W., Toney, M. D., and Shen, B. (2003) J. Am. Chem. Soc. 125, 6062-6063]. Here, we present a thorough analysis of the properties of SgcC4. L-Tyrosine is the best substrate among those tested and most likely serves as the in vivo precursor for the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety. The presence of MIO in the active site is supported by several lines of evidence. (1) Addition of ATP or divalent metal ions has no effect on its aminomutase activity. (2) SgcC4 has optimal activity at pH ∼8.8, similar to the pH optima of MIO-dependent ammonia lyases. (3) SgcC4 is strongly inhibited by sodium borohydride and potassium cyanide, but preincubation with L-tyrosine or 4-hydroxycinnamate largely prevents this inhibition. (4) The difference spectrum between SgcC4 and its S153A mutant shows a positive peak at ∼310 nm, indicative of MIO. (5) The S153A mutation lowers kcat/K M 640-fold. The SgcC4-catalyzed conversion of L-tyrosine to (S)-β-tyrosine proceeds via 4-hydroxycinnamate as an intermediate. The latter also acts as a competitive inhibitor with respect to L-tyrosine and serves as an alternative substrate for the production of β-tyrosine in the presence of an amino source. A full time course for the SgcC4-catalyzed interconversion between L-tyrosine, β-tyrosine, and 4-hydroxycinnamate was measured and analyzed to provide estimates for the rate constants in a minimal mechanism. SgcC4 also exhibits a β-tyrosine racemase activity, but α-tyrosine racemase activity was not detected.

Original languageEnglish (US)
Pages (from-to)12708-12718
Number of pages11
JournalBiochemistry
Volume42
Issue number43
DOIs
StatePublished - Nov 4 2003

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Enediynes
Biosynthesis
Tyrosine
Anti-Bacterial Agents
Kinetics
Racemases and Epimerases
Phenylalanine
C 1027
4-methylideneimidazole-5-one
Ammonia-Lyases
Catalytic Domain
Sodium Cyanide
Potassium Cyanide
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic Analysis of the 4-Methylideneimidazole-5-one-Containing Tyrosine Aminomutase in Enediyne Antitumor Antibiotic C-1027 Biosynthesis. / Christenson, Steven D.; Wu, Weiming; Spies, M. Ashley; Shen, Ben; Toney, Michael D.

In: Biochemistry, Vol. 42, No. 43, 04.11.2003, p. 12708-12718.

Research output: Contribution to journalArticle

Christenson, Steven D. ; Wu, Weiming ; Spies, M. Ashley ; Shen, Ben ; Toney, Michael D. / Kinetic Analysis of the 4-Methylideneimidazole-5-one-Containing Tyrosine Aminomutase in Enediyne Antitumor Antibiotic C-1027 Biosynthesis. In: Biochemistry. 2003 ; Vol. 42, No. 43. pp. 12708-12718.
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abstract = "The enediyne antitumor antibiotic C-1027 contains an unusual (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety, which requires an aminomutase for its biosynthesis. Previously, we established that SgcC4 is an aminomutase that catalyzes the conversion of L-tyrosine to (S)-β-tyrosine and employs 4-methylideneimidazole-5-one (MIO) at its active site [Christenson, S. D., Liu, W., Toney, M. D., and Shen, B. (2003) J. Am. Chem. Soc. 125, 6062-6063]. Here, we present a thorough analysis of the properties of SgcC4. L-Tyrosine is the best substrate among those tested and most likely serves as the in vivo precursor for the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety. The presence of MIO in the active site is supported by several lines of evidence. (1) Addition of ATP or divalent metal ions has no effect on its aminomutase activity. (2) SgcC4 has optimal activity at pH ∼8.8, similar to the pH optima of MIO-dependent ammonia lyases. (3) SgcC4 is strongly inhibited by sodium borohydride and potassium cyanide, but preincubation with L-tyrosine or 4-hydroxycinnamate largely prevents this inhibition. (4) The difference spectrum between SgcC4 and its S153A mutant shows a positive peak at ∼310 nm, indicative of MIO. (5) The S153A mutation lowers kcat/K M 640-fold. The SgcC4-catalyzed conversion of L-tyrosine to (S)-β-tyrosine proceeds via 4-hydroxycinnamate as an intermediate. The latter also acts as a competitive inhibitor with respect to L-tyrosine and serves as an alternative substrate for the production of β-tyrosine in the presence of an amino source. A full time course for the SgcC4-catalyzed interconversion between L-tyrosine, β-tyrosine, and 4-hydroxycinnamate was measured and analyzed to provide estimates for the rate constants in a minimal mechanism. SgcC4 also exhibits a β-tyrosine racemase activity, but α-tyrosine racemase activity was not detected.",
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AU - Shen, Ben

AU - Toney, Michael D.

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N2 - The enediyne antitumor antibiotic C-1027 contains an unusual (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety, which requires an aminomutase for its biosynthesis. Previously, we established that SgcC4 is an aminomutase that catalyzes the conversion of L-tyrosine to (S)-β-tyrosine and employs 4-methylideneimidazole-5-one (MIO) at its active site [Christenson, S. D., Liu, W., Toney, M. D., and Shen, B. (2003) J. Am. Chem. Soc. 125, 6062-6063]. Here, we present a thorough analysis of the properties of SgcC4. L-Tyrosine is the best substrate among those tested and most likely serves as the in vivo precursor for the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety. The presence of MIO in the active site is supported by several lines of evidence. (1) Addition of ATP or divalent metal ions has no effect on its aminomutase activity. (2) SgcC4 has optimal activity at pH ∼8.8, similar to the pH optima of MIO-dependent ammonia lyases. (3) SgcC4 is strongly inhibited by sodium borohydride and potassium cyanide, but preincubation with L-tyrosine or 4-hydroxycinnamate largely prevents this inhibition. (4) The difference spectrum between SgcC4 and its S153A mutant shows a positive peak at ∼310 nm, indicative of MIO. (5) The S153A mutation lowers kcat/K M 640-fold. The SgcC4-catalyzed conversion of L-tyrosine to (S)-β-tyrosine proceeds via 4-hydroxycinnamate as an intermediate. The latter also acts as a competitive inhibitor with respect to L-tyrosine and serves as an alternative substrate for the production of β-tyrosine in the presence of an amino source. A full time course for the SgcC4-catalyzed interconversion between L-tyrosine, β-tyrosine, and 4-hydroxycinnamate was measured and analyzed to provide estimates for the rate constants in a minimal mechanism. SgcC4 also exhibits a β-tyrosine racemase activity, but α-tyrosine racemase activity was not detected.

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