title: Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism.
abstract: In plant secondary metabolism, beta-acetal ester-dependent acyltransferases, such as the 1-O-sinapoyl-beta-glucose:l-malate sinapoyltransferase (SMT; EC 2.3.1.92), 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 increase, resulting in an overall 3900-fold activity compared to the AtSMT cDNA of plant origin. Detailed kinetic analyses of the recombinant protein indicated a random sequential bi-bi mechanism for the SMT-catalyzed transacylation, in contrast to a double displacement (ping-pong) mechanism, characteristic of serine carboxypeptidases.
authors.affiliation: Department of Secondary Metabolism, Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Stehle
authors.forename: Milton T
authors.initials: MT
authors.lastname: Stubbs
authors.forename: Dieter
authors.initials: D
authors.lastname: Strack
authors.forename: Carsten
authors.initials: C
authors.lastname: Milkowski
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008293:Malates / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018411:Spodoptera; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D008293:Malates; D011994:Recombinant Proteins; D000217:Acyltransferases; D002268:Carboxypeptidases; C022633:serine carboxypeptidase