A small membrane-peripheral region close to the active center determines regioselectivity of membrane-bound fatty acid desaturases from Aspergillus nidulans.
Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Delta12 desaturase and a processive bifunctional oleoyl-Delta12/linoleoyl-omega3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Delta12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Delta12/omega3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.
Hoffmann
Mareike
M
Abteilung Biochemie der Pflanze, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Hornung
Ellen
E
Abteilung Biochemie der Pflanze, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Busch
Silke
S
Abteilung Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, Grisebachstr. 8, D-37077 Göttingen, Germany.
Kassner
Nina
N
Abteilung Biochemie der Pflanze, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Ternes
Philipp
P
Faculteit Scheikunde, Universiteit Utrecht, H. R. Kruytgebouw N601, Padualaan 8, NL-3584 CH Utrecht, Netherlands.
Braus
Gerhard H
GH
Abteilung Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, Grisebachstr. 8, D-37077 Göttingen, Germany.
Feussner
Ivo
I
Abteilung Biochemie der Pflanze, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany. Electronic address: ifeussn@gwdg.de.
D001233:Aspergillus nidulans / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D044943:Fatty Acid Desaturases