TAILIEUCHUNG - Báo cáo khoa học: Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism

In plant secondary metabolism, b-acetal ester-dependent acyltransferases, such as the 1-O-sinapoyl-b-glucose:l-malate sinapoyltransferase (SMT; EC ), are homologous to serine carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT (AtSMT) have predicted amino acid residues involved in substrate recognition and catalysis, confirming the main functional elements conserved within the serine carboxypeptidase pro-tein family. | ỊFEBS Journal Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism Felix Stehle1 Milton T. Stubbs2 Dieter Strack1 and Carsten Milkowski1 1 Department of Secondary Metabolism Leibniz Institute of Plant Biochemistry IPB Halle Saale Germany 2 Institute of Biochemistry and Biotechnology Martin-Luther-University Halle-Wittenberg Germany Keywords acyltransferase enzymatic kinetic mechanism heterologous expression molecular evolution serine carboxypeptidase-like proteins Correspondence D. Strack Department of Secondary Metabolism Leibniz Institute of Plant Biochemistry IPB Weinberg 3 06120 Halle Saale Germany Fax 49 345 5582 1509 Tel 49 345 5582 1500 E-mail Received 13 November 2007 revised 13 December 2007 accepted 14 December 2007 doi In plant secondary metabolism b-acetal ester-dependent acyltransferases such as the 1-O-sinapoyl-b-glucose L-malate sinapoyltransferase SMT EC are homologous to serine carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT AtSMT have predicted amino acid residues involved in substrate recognition and catalysis confirming the main functional elements conserved within the serine carboxypeptidase protein family. However the functional shift from hydrolytic to acyltransferase activity and structure-function relationship of AtSMT remain obscure. To address these questions a heterologous expression system for AtSMT has been developed that relies on Saccharomyces cerevisiae and an episomal leu2-d vector. Codon usage adaptation of AtSMT cDNA raised the produced SMT activity by a factor of approximately three. N-terminal fusion to the leader peptide from yeast proteinase A and transfer of this expression cassette to a high copy vector led to further increase in SMT expression by factors of 12 and 42 respectively. Finally upscaling the biomass production by fermenter cultivation lead to another 90-fold .

• Báo cáo khoa học
• Report medicine
• traditional medicine
• scientific reports
• oxidation
• chemical reaction
• tumor cells
• medical knowledge
• medical research
• analysis of cytoplasmic
• Crystal structure
• bacteria
• hemoglobin
• medicine
• cell proliferation
• breast cancer
• gastric cancer
• hormone medicine
• biochemical studies
• environmental medicine