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
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
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.