Crystallographic studies and preliminary X-ray investigation of (S)-p-hydroxy-mandelonitrile lyase fromSorghum bicolor(L.)

Abstract

Abstract(S)-p-Hydroxy-mandelonitrile lyase from Sorghum bicolor has been crystallized in three different forms using the hangingdrop vapor-diffusion technique. Crystal tbrm I is obtained from 1.4M (NH4)2SO 4 in 100mM Na-acetate, pH 4.6, and belongs to the orthorhombic space group P2~2t2 ~. The cell dimensions are a = 71.4, b = 95.8, c = 149.1 A. A complete set of diffraction data has been collected to 2.6 A resolution. Form II crystals are grown from 500mM Li2SO 4 in 13% polyethylene glycol 8000. These crystals appear as hexagonal plates and diffract to 2.98]k resolution but apparently are twinned. Cocrystallizing hydroxynitrile lyase with the inhibitor benzoic acid using 1.4M (NH4)2SO 4 in 100mM Na citrate, pH 5.4 as precipitant yields crystal form III, which belongs to the monoclinic space group C2 with a = 150.7, b= 103.7, c=90.6A, fl= 101.3. X-ray diffraction data were collected to 2.3 A resolution.

Cyanohydrins in Nature and the Laboratory: Biology, Preparations, and Synthetic Applications

“…65 Preliminary X-ray diffraction studies have been published for crystals of PaHNL, 66 MeHNL, 67 and SbHNL. 68 X-ray diffraction studies on crystals of HbHNL have now enabled the solution of the first three-dimensional structure of an HNL: 69,70 in addition, mutagenesis studies 63,71 are consistent with the assignment of HbHNL to the R/β-hydrolase fold family, which has implications for the enzyme mechanism (Section III.F).…”

“…cDNA cloning has been carried out for five genes, and recombinant enzymes have been obtained in three cases; namely, MeHNL (overexpressed in Escherichia coli ), , LuHNL (in E. coli and, optimally, Pichia pastoris ), , and HbHNL (in E. coli , Saccharomyces cerevisiae , and, optimally, P. pastoris ). , Difficulties have been encountered with SbHNL due to complex posttranslational processing of the native enzyme . Preliminary X-ray diffraction studies have been published for crystals of PaHNL, MeHNL, and SbHNL . X-ray diffraction studies on crystals of HbHNL have now enabled the solution of the first three-dimensional structure of an HNL: 69,70 in addition, mutagenesis studies 63,71 are consistent with the assignment of HbHNL to the α/β-hydrolase fold family, which has implications for the enzyme mechanism (Section III.F).…”

Crystal Structure of Hydroxynitrile Lyase from Sorghum bicolor in Complex with the Inhibitor Benzoic Acid: A Novel Cyanogenic Enzyme,

“…We recently crystallized the SbHNL isoenzyme II (SbHNL-II) in the presence of the inhibitor benzoic acid (14) and have now determined the three-dimensional structure to 2.3 Å resolution. With the structure presented here, we have examined the relationship of the modified SbHNL-II active site sequence motif to the enzyme's overall three-dimensional structure.…”

Screening for New Hydroxynitrilases from Plants

“…[23][24][25][26] As described here, we developed a new method for measurement of mandelonitrile lyase activity by detecting mandelonitirile formation with HPLC. To assay the enzyme activity, two methods has been used.…”