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

Yasuhisa Asano,Makoto Nakabayashi,Daijun Zheng

doi:10.1016/j.jbc.2022.101650

Yasuhisa Asano, Makoto Nakabayashi + Show 1 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101650

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Abstract

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.

Highlights

Hydroxynitrile lyases (HNLs) are primarily found in higher plants [1,2], microorganisms [3,4,5], and millipedes [6,7] as crucial enzymes participating in the process of cyanogenesis, in which it was identified to catalyze the decomposition of cyanohydrins to corresponding carbonyl compounds and toxic hydrogen cyanide (HCN) [8,9,10]
The sequence alignment of LuHNL suggests that its structure is closer to that of Zn2+-containing alcohol dehydrogenase (ADH), sharing the substrate and NAD+ binding domains
Absence of ADH activity in LuHNL and vice versa In horse liver ADH (Hl_ADH), the hydroxyl group of the substrate was bonded to the catalytic Zn2+ that coordinated with Cys46, Journal Pre-proof His67, and Cys174 (Fig. 8A)

Summary

Introduction

Hydroxynitrile lyases (HNLs) are primarily found in higher plants [1,2], microorganisms [3,4,5], and millipedes [6,7] as crucial enzymes participating in the process of cyanogenesis, in which it was identified to catalyze the decomposition of cyanohydrins to corresponding carbonyl compounds and toxic hydrogen cyanide (HCN) [8,9,10]. The subsequent site-directed mutagenesis analysis of LuHNL by its overexpression in Pichia pastoris indicated that the residues involved in catalysis of Zn2+-ADHs were functionally important in LuHNL From these results, it was presumed that all the Zn2+ ions in LuHNL possess only the function of stabilizing the structure and not participating in the catalysis [46]. Three structures of LuHNL were determined: ligandfree LuHNL (LuHNL_lig_free, PDB ID: 7VB3), acetone cyanohydrin-complexed LuHNL (LuHNL_CNH, PDB ID: 7VB5), and (R)-2-butanone cyanohydrin-complexed LuHNL (LuHNL_BCN, PDB ID: 7VB6) Based on these crystal structures and site-directed mutagenesis analysis results, we proposed a catalytic mechanism for LuHNL on cyanohydrin decomposition and elucidated the function of NAD+ and Zn2+ in LuHNL.

Results

Discussion

Experimental procedures

Declaration of interests