Separation by high-performance liquid chromatography of α- and β-amino acids: Application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase

Dj, Aberhart

doi:10.1016/0003-2697(88)90294-1

Cited by 14 publications

(3 citation statements)

References 25 publications

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“…HPLC analysis of reaction products employed precolumn derivatization of amino acids with OPA. The resulting isoindole improves the resolution of the Rand β-isomers of various amino acids, including tyrosine (32). OPA derivatives of reaction samples were made by combining the neutralized filtrates of SgcC4 reactions with an equal volume of OPA reagent for 1 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2003

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The enediyne antitumor antibiotic C-1027 contains an unusual (S)-3-chloro-4,5-dihydroxy-beta-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)-beta-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-beta-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 approximately 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 approximately 310 nm, indicative of MIO. (5) The S153A mutation lowers k(cat)/K(M) 640-fold. The SgcC4-catalyzed conversion of L-tyrosine to (S)-beta-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 beta-tyrosine in the presence of an amino source. A full time course for the SgcC4-catalyzed interconversion between L-tyrosine, beta-tyrosine, and 4-hydroxycinnamate was measured and analyzed to provide estimates for the rate constants in a minimal mechanism. SgcC4 also exhibits a beta-tyrosine racemase activity, but alpha-tyrosine racemase activity was not detected.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2003

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“…The reaction was terminated by addition of HCl to pH < 2. The quenched mixture was derivatized with ophthaldialdehyde 11 and subjected to HPLC analysis. 12 Baseline separation for 3 and 4 as o-phthaldialdehyde derivatives and 5 was achieved (Figure 3, inset).…”

mentioning

confidence: 99%

A Novel 4-Methylideneimidazole-5-one-Containing Tyrosine Aminomutase in Enediyne Antitumor Antibiotic C-1027 Biosynthesis

et al. 2003

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The C-1027 enediyne antibiotic contains an unusual 3-chloro-4,5-dihydroxy-β-phenylalanine moiety that is thought to be derived from tyrosine by an aminomutase reaction. However, none of the genes identified within the C-1027 gene cluster encode proteins with strong homology to known aminomutases. The sgcC4 gene encodes a protein with strong homology to dehydroalanine-dependent histidine/phenylalanine ammonia lyases. The sgcC4 gene was expressed in E. coli, and overproduced SgcC4 was purified as a His6-tagged fusion protein. Biochemical characterization of the purified SgcC4 establishes 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. The latter was supported by borohydride and cyanide inhibition studies and confirmed by site-directed mutagenesis. The S153A mutant exhibited a 340-fold decrease in k cat/K M. SgcC4 represents a novel type of aminomutase, extending the known MIO chemistry from ammonia lyases into aminomutases.

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“…A non-cobalamine-dependent 2,3-aminomutase activity converting leucine to (R)-b-leucine (31) has been detected in the plant Andrographis paniculata [93], but no further reliable reports on the occurrence of b-leucine are known so far [94].…”

Section: B-leucinementioning

confidence: 99%

β‐Amino Acid Biosynthesis

Spiteller

2009

Amino Acids, Peptides and Proteins in Organic Chemistry

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Separation by high-performance liquid chromatography of α- and β-amino acids: Application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase

Cited by 14 publications

References 25 publications

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

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

A Novel 4-Methylideneimidazole-5-one-Containing Tyrosine Aminomutase in Enediyne Antitumor Antibiotic C-1027 Biosynthesis

β‐Amino Acid Biosynthesis

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