2-Amino-3-ketobutyrate CoA ligase （KBL） of Escherichia coli is a member of the a-oxoamine synthase family; it catalyzes the condensation reaction between glycine and acetyl CoA to yield 2-amino-3-ketobutyrate. We ha...2-Amino-3-ketobutyrate CoA ligase （KBL） of Escherichia coli is a member of the a-oxoamine synthase family; it catalyzes the condensation reaction between glycine and acetyl CoA to yield 2-amino-3-ketobutyrate. We have previously shown that KBL catalyzes the exchange ofpro-R hydrogen of glycine with protons in the medium; however, the kinetics of this reaction has never been determined. In this study, we calculated the kinetic parameters of this exchange reaction by using different concentrations of [2RS- 3H2： 2-14C] glycine. The rate of the exchange reaction was determined by measuring the 3H/14C ratio in recovered ｜2S- 3H： 2J4C]glycine. The Lineweaver-Burk plot showed that Km and kcat of this reaction were 3.8 ×10^-3 M and 0.22 S^-11, respectively. On the other hand, Km and kcat values of the overall KBL-mediated catalysis were correspondingly 1.23 × 10^-2 M and 1.19 S^-1. Thus, the rate of the exchange reaction was almost five times lower than that of overall KBL catalysis.展开更多
An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and ...An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.展开更多
Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop （Humulus lupulus） glandular trichomes. Branched short-chain acyI-CoAs （e.g. isobutyry...Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop （Humulus lupulus） glandular trichomes. Branched short-chain acyI-CoAs （e.g. isobutyryI-CoA, isovaleryl- CoA and 2-methylbutyryI-CoA）, derived from the degradation of branched-chain amino acids （BCAAs）, are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyI-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HICCL2 （carboxyl CoA ligase） protein had high specific activity for isovaleric acid as a substrate （Kcat/Km = 4100 s-~ M-l）, whereas recombinant HICCL4 specifically utilized isobutyric acid （Kcat/Km = 1800 s-1 M-1） and 2-methylbutyric acid （Kcat/ Km = 6900 s-1 M-~） as substrates. Both HICCLs, like hop valerophenone synthase （HIVPS）, were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HICCL2 and HICCL4 with HIVPS in yeast led to significant production of acylphloroglucinols （the direct precursors for bitter acid biosynthesis）, which further confirmed the biochemical function of these two HICCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid：CoA ligases in planta are also discussed.展开更多
文摘2-Amino-3-ketobutyrate CoA ligase （KBL） of Escherichia coli is a member of the a-oxoamine synthase family; it catalyzes the condensation reaction between glycine and acetyl CoA to yield 2-amino-3-ketobutyrate. We have previously shown that KBL catalyzes the exchange ofpro-R hydrogen of glycine with protons in the medium; however, the kinetics of this reaction has never been determined. In this study, we calculated the kinetic parameters of this exchange reaction by using different concentrations of [2RS- 3H2： 2-14C] glycine. The rate of the exchange reaction was determined by measuring the 3H/14C ratio in recovered ｜2S- 3H： 2J4C]glycine. The Lineweaver-Burk plot showed that Km and kcat of this reaction were 3.8 ×10^-3 M and 0.22 S^-11, respectively. On the other hand, Km and kcat values of the overall KBL-mediated catalysis were correspondingly 1.23 × 10^-2 M and 1.19 S^-1. Thus, the rate of the exchange reaction was almost five times lower than that of overall KBL catalysis.
文摘An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.
基金the National Program on Key Basic Research Projects，the 'One hundred talents' project of the Chinese Academy of Sciences，the National Natural Sciences Foundation of China，the National Science Foundation，the State Key Laboratory of Plant Genomics of China
文摘Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop （Humulus lupulus） glandular trichomes. Branched short-chain acyI-CoAs （e.g. isobutyryI-CoA, isovaleryl- CoA and 2-methylbutyryI-CoA）, derived from the degradation of branched-chain amino acids （BCAAs）, are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyI-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HICCL2 （carboxyl CoA ligase） protein had high specific activity for isovaleric acid as a substrate （Kcat/Km = 4100 s-~ M-l）, whereas recombinant HICCL4 specifically utilized isobutyric acid （Kcat/Km = 1800 s-1 M-1） and 2-methylbutyric acid （Kcat/ Km = 6900 s-1 M-~） as substrates. Both HICCLs, like hop valerophenone synthase （HIVPS）, were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HICCL2 and HICCL4 with HIVPS in yeast led to significant production of acylphloroglucinols （the direct precursors for bitter acid biosynthesis）, which further confirmed the biochemical function of these two HICCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid：CoA ligases in planta are also discussed.
