Structural characterization of Linum usitatissimum hydroxynitrile lyase: A new cyanohydrin decomposition mechanism involving a cyano-zinc complex

• Published in: The Journal of biological chemistry Vol. 298; no. 3; p. 101650
• Main Authors: Zheng, Daijun, Nakabayashi, Makoto, Asano, Yasuhisa
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2022; American Society for Biochemistry and Molecular Biology
• Subjects: Aldehyde-Lyases - chemistry; Aldehyde-Lyases - metabolism; Binding Sites; Crystallography, X-Ray; enzyme mechanism; enzyme structure; Flax - enzymology; hydroxynitrile lyase; Models, Molecular; NAD - metabolism; nicotinamide adenine dinucleotide (NAD); Plant Proteins - chemistry; Plant Proteins - metabolism; X-ray crystallography; zinc; Zinc - chemistry; Zinc - metabolism; X-ray crystallography; CV; nicotinamide adenine dinucleotide (NAD); ADH; HNL; LuHNL; enzyme mechanism; BCN; KPB; zinc; enzyme structure; hydroxynitrile lyase
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2022.101650

Hydroxynitrile lyase from Linum usitatissimum (LuHNL) is an enzyme involved in the catabolism of cyanogenic glycosides to release hydrogen cyanide upon tissue damage. This enzyme strictly conserves the substrate- and NAD(H)-binding domains of Zn2+-containing alcohol dehydrogenase (ADH); however, there is no evidence suggesting that LuHNL possesses ADH activity. Herein, we determined the ligand-free 3D structure of LuHNL and its complex with acetone cyanohydrin and (R)-2-butanone cyanohydrin using X-ray crystallography. These structures reveal that an A-form NAD+ is tightly but not covalently bound to each subunit of LuHNL. The restricted movement of the NAD+ molecule is due to the “sandwich structure” on the adenine moiety of NAD+. Moreover, the structures and mutagenesis analysis reveal a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H2O/Lys162 of LuHNL. The deprotonated Lys162 and protonated Glu323 residues are presumably stabilized by a partially desolvated microenvironment. In summary, the substrate binding geometry of LuHNL provides insights into the differences in activities of LuHNL and ADH, and identifying this novel reaction mechanism is an important contribution to the study of hydroxynitrile lyases.