title: Structures of almond hydroxynitrile lyase isoenzyme 5 provide a rationale for the lack of oxidoreductase activity in flavin dependent HNLs.
abstract: Hydroxynitrile lyases (HNLs) catalyze the asymmetric addition of HCN to aldehydes producing enantiomerically pure cyanohydrins. These enzymes can be heterologously expressed in large quantities making them interesting candidates for industrial applications. The HNLs from Rosaceae evolved from flavin dependent dehydrogenase/oxidase structures. Here we report the high resolution X-ray structure of the highly glycosylated Prunus amygdalus HNL isoenzyme5 (PaHNL5 V317A) expressed in Aspergillus niger and its complex with benzyl alcohol. A comparison with the structure of isoenzyme PaHNL1 indicates a higher accessibility to the active site and a larger cavity for PaHNL5. Additionally, the PaHNL5 complex structure with benzyl alcohol was compared with the structurally related aryl-alcohol oxidase (AAO). Even though both enzymes contain an FAD-cofactor and histidine residues at crucial positions in the active site, PaHNL5 lacks the oxidoreductase activity. The structures indicate that in PaHNLs benzyl alcohol is bound too far away from the FAD cofactor in order to be oxidized.
authors.affiliation: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50/3, 8010 Graz, Austria.
authors.forename: Tea
authors.initials: T
authors.lastname: Pavkov-Keller
authors.affiliation: DSM, Alexander Flemingplaan 1, 2613 AX Delft, Netherlands.
authors.forename: Janny
authors.initials: J
authors.lastname: Bakhuis
authors.affiliation: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50/3, 8010 Graz, Austria.
authors.forename: Georg
authors.initials: G
authors.lastname: Steinkellner
authors.affiliation: DSM, Alexander Flemingplaan 1, 2613 AX Delft, Netherlands.
authors.forename: Fenneke
authors.initials: F
authors.lastname: Jolink
authors.affiliation: DSM, Alexander Flemingplaan 1, 2613 AX Delft, Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: Keijmel
authors.affiliation: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria; Research Unit Functional Proteomics and Metabolic Pathways, Institute of Pathology, Medical University of Graz, Omics Center Graz, BioTechMed-Graz, Stiftingtalstrasse 24, 8010 Graz, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.affiliation: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50/3, 8010 Graz, Austria. Electronic address: karl.gruber@uni-graz.at.
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D005415:Flavins / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D000068181:Prunus dulcis / Q000201:enzymology*
chemical_list: D005415:Flavins; D007527:Isoenzymes; D010940:Plant Proteins; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase
keywords: Benzyl alcohol; Hydroxynitrile lyase; Prunus amygdalus; X-ray structure