Expression of the Zn2+-containing hydroxynitrile lyase from flax (Linum usitatissimum) in Pichia pastoris - utilization of the recombinant enzyme forenzymatic analysis and site-directed mutagenesis

Citation
K. Trummler et al., Expression of the Zn2+-containing hydroxynitrile lyase from flax (Linum usitatissimum) in Pichia pastoris - utilization of the recombinant enzyme forenzymatic analysis and site-directed mutagenesis, PLANT SCI, 139(1), 1998, pp. 19-27
Citations number
23
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT SCIENCE
ISSN journal
01689452 → ACNP
Volume
139
Issue
1
Year of publication
1998
Pages
19 - 27
Database
ISI
SICI code
0168-9452(199812)139:1<19:EOTZHL>2.0.ZU;2-8
Abstract
Hydroxynitrile lyases (HNL) are involved in the catabolism of cyanogenic gl ycosides in cyanogenic plants and are powerful tools in the stereoselective synthesis of cyanohydrins. The recent cloning of the hydroxynitrile lyase from flax (Linum usitatissimum: LuHNL) reveals that this enzyme defines a n ovel class of HNL. Thorough biochemical and mutational analysis of LuHNL ha ve been hampered by low expression levels of the recombinant enzyme in Esch erichia coli. To overcome this impediment, we have cloned a myc-His-tagged LuHNL-cDNA under control of the methanol-inducible AOX1 (alcohol oxidase) p romotor of Pichia pastoris and introduced it in the SMD1168 strain. Recombi nant LuHNL was kinetically indistinguishable from the authentic flax enzyme . Immobilized LuHNL was used for synthesis of several aliphatic (R)-cyanohy drins in a preparative scale to analyze the products according to enantiome ric excess and yield of reaction. LuHNL has significant homologies to membe rs of the Zn2+-containing alcohol dehydrogenases (Zn2+-ADHs). In particular , residues responsible for coordination of Zn2+ ions or fulfilling structur al or functional tasks in Zn2+-ADHs are conserved. We found about 2-4 mol z inc per mol of recombinant LuHNL using atom absorption spectroscopy in a no n His-lagged version of LuHNL. Using site-directed mutagenesis, we substitu ted several of the conserved residues against alanine in LuHNL and found th at in most cases, HNL-activity was impaired. Hence, it seems that LuHNL and Zn2+-ADHs have similar structural requirements with respect to maintaining a catalytically active structure. Residues essentially involved in catalys is of Zn2+-ADHs are also of functional importance in LuHNL, suggesting that the removal of the proton from alcohol and cleavage of cyanohydrins can be fulfilled by similar active site structures. (C) 1998 Elsevier Science Ire land Ltd. All rights reserved.