Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Abstract

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

The crystal structure and catalytic mechanism of hydroxynitrile lyase from passion fruit, Passiflora edulis

“…The interactions of the nitrile group of ( R )‐MAN in Pe HNL resembles to those in Dt HNL . The protonation of both Glu54 in Pe HNL and Asp in Dt HNL was proposed . It has been known that the protonation of the carboxyl group is stabilized in the hydrophobic environment by excluding water molecules donating a proton .…”

Production of Hydroxynitrile Lyase from Davallia tyermannii (DtHNL) in Komagataella phaffii and Its Immobilization as a CLEA to Generate a Robust Biocatalyst

“…This activity was obtained with the cell‐free lysate, without any purification or concentration steps. This activity indicates that Dt HNL1 constitutes approximately 80 % of the total protein content (standard values for the specific activity of purified Dt HNL1 were 320–350 U mg −1 with a 20 % confidence interval). The high Dt HNL1 level is further confirmed by gel electrophoresis (Figure B).…”

“…It is a small (20.1 kDa), dimeric protein, with high activity for ( R )‐mandelonitrile cleavage ( k cat =144 s −1 ). Moreover, Dt HNL1 can convert different aldehydes into the respective cyanohydrins with high yields and ee values . Therefore, Dt HNL1 is a powerful biocatalyst for enantiomerically pure cyanohydrin production.…”

“…Therefore, Dt HNL1 is a powerful biocatalyst for enantiomerically pure cyanohydrin production. The enzyme is active under acidic conditions; however, it is not sufficiently stable under process conditions and its activity constantly decreases at pH 2.5 …”

Stabilization of Hydroxynitrile Lyases from Two Variants of Passion Fruit, Passiflora edulis Sims and Passiflora edulis Forma flavicarpa, by C‐Terminal Truncation

“…HNLs have mainly been discovered and characterized from cyanogenic plants; these include the R ‐selective ones derived from Prunus amygdalus ( Pa HNL), Prunus serotina Ehrh. ( Ps HNL), Prunus mume ( Pm HNL), Prunus armeniaca L. ( Pars HNL), Prunus communis ( Pc HNL), Linum usitatissimum ( Lu HNL), Phlebodium aureum ( Pha HNL), Arabidopsis thaliana ( At HNL), Eriobotrya japonica ( Ej HNL), Passiflora edulis forma flavicarpa ( Pe HNL), Davallia tyermannii ( Dt HNL), and Nandina domestica ( Nd HNL), and the S ‐selective ones derived from Hevea brasiliensis ( Hb HNL), Manihot esculenta ( Me HNL), Sorghum bicolor ( Sb HNL), and Baliospermum montanum ( Bm HNL) . Among the HNLs of plant origin, Sb HNL, Ej HNL, Pe HNL, Pa HNL, Pc HNL, and Pm HNL are glycosylated proteins, whereas, Lu HNL, Bm HNL, At HNL, Nd HNL, Hb HNL, Me HNL, and Pha HNL are not glycosylated.…”