Structure-function relationships of SCPL acyltransferases
CARSTEN MILKOWSKI
Friedrich-Schiller-Universität Jena
Institut für Allgemeine Botanik
Am Planetarium 1
07743 Jena
Carsten.Milkowski@uni-jena.de
http://www.uni-jena.de/Institut.html
DIETER STRACK
Leibniz Institute of Plant Biochemistry
Dep. Secondary Metabolism
Weinberg 3
D-06120 Halle (Saale)
Dieter.Strack@ipb-halle.de
http://www.ipb-halle.de/en/research/secondary-metabolism/
MILTON T. STUBBS
Department of Physical Biotechnology
Martin Luther University
Kurt-Mothes-Str. 3
D-06120 Halle (Saale)
stubbs@biochemtech.uni-halle.de
http://www.biochemtech.uni-halle.de/biotechnologie/xray/
In plant phenylpropanoid metabolism acyltransferases were found which accept energy-rich hydroxycinnamoyl glucose esters (beta acetal esters) as acyl donors instead of CoA activated thioesters. Sequence features indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. Belonging to the highly diverse group of alpha/beta hydrolase fold enzymes, SCPL acyltransferases apparently make use of a catalytic triad formed by a seryl residue as nucleophile, an aspartyl and histidyl residue.
A gene encoding the enzyme of interest, 1-O-sinapoylglucose:malate sinapoyltransferase (SMT, EC 2.3.1.92) from Arabidopsis, had been identified previously. However functional analysis of the cloned cDNA was basically confined to mutant complementation. As our work aimed at unravelling the mechanistic aspects which drive the functional shift from hydrolase to acyltransferase activity requires structure elucidation, we had to develop a robust heterologous expression system for this enzyme class. Neither expression in E. coli followed by in vitro refolding nor SMT expression in tobacco tissue or cell culture has proven successful. Only in S. cerevisae a very low SMT expression was observed. Codon usage optimization and signal sequence alteration were shown to improve the expression by a factor of 150. Unfortunately, the expression level could never been increased over 100 µg/l culture. Hence, we established fermentation to raise the protein yield to a level appropriate for enzyme kinetics and crystallization trials.
Additionally, initial structural modeling studies of SMT were performed. Accompanying site-directed mutagenesis examinations helped to identify sequence elements of functional importance.
In order to augment sequence information for future evolutionary analyses, homology-based cloning approaches are used to isolate cDNAs coding for related acyltransferases.
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