Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

O-Glycoside hydrolases (EC 3.2.1.x) constitute a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. These enzymes have been classified into distinct families based on amino acid sequence similarities (Henrissat, 1991; Henrissat and Bairoch, 1996). One of these, family 1, includes many β-glucosidases (EC 3.2.1.21) that play diverse and important roles in prokaryotes and eukaryotes. In bacteria and fungi, the cellulolytic β-glucosidases are key enzymes in biomass conversion (Béguin, 1990; Fowler, 1993), whereas in animals, lysosomal glucocerebrosidase, for example, is critical to glycosphingolipid metabolism (Grabowski et al., 1993). In higher plants, β-glucosidases have been implicated in such fundamental processes as chemical defense against herbivores and pathogens (Conn, 1979), lignification (Dharmawardhana et al., 1995), and regulation of the biological activity of phytohormones by hydrolysis of their inactive hormone-glucoside conjugates (Falk and Rask, 1995).

In recent years, our laboratory has been investigating the nature and regulation of β-glucosidases involved in large-scale cyanogenesis (HCN production) in plants. As an experimental system, we have used black cherry (Prunus serotina Ehrh.), a species grown for its fruit and high-value hardwood. Like other rosaceous stone fruits, black cherry accumulates the cyanogenic monoglucoside (R)-prunasin [the O-β-d-glucoside of (R)-mandelonitrile] in its leaves and immature fruits (Kingsbury, 1964). The related diglucoside (R)-amygdalin [the β-gentiobioside of (R)-mandelonitrile] is found in high concentrations in mature seeds of these crops. Upon seed disruption, amygdalin is hydrolyzed to mandelonitrile by a two-step process. First, amygdalin hydrolase (AH; EC 3.2.1.117) hydrolyzes the β-1,6-glycosidic bond of amygdalin, yielding prunasin and d-Glc. Prunasin is then hydrolyzed to mandelonitrile and d-Glc by prunasin hydrolase (PH; EC 3.2.1.118). Because AH and PH show very pronounced specificities toward their respective glucosidic substrates (Kuroki and Poulton, 1986, 1987), both enzymes are required in cherry macerates for complete hydrolysis of amygdalin. Finally, mandelonitrile dissociates either enzymatically in the presence of the α-hydroxynitrile lyase mandelonitrile lyase (MDL; EC 4.1.2.10) or spontaneously, generating HCN and benzaldehyde (Hu and Poulton, 1999). In disrupted black cherry leaves, prunasin is degraded to HCN in similar fashion by PH and MDL.

In previous studies, AH and PH were purified to homogeneity from mature seeds, and their major physicochemical properties were characterized (Poulton, 1993). Subsequently, we demonstrated by immunocytochemistry and tissue printing that tissue level compartmentation prevents premature cyanogenesis in undamaged seeds. Whereas amygdalin accumulates in cotyledonary parenchyma cells, AH and PH are located in the procambium, where each β-glucosidase occurs as multiple forms (Li et al., 1992; Swain et al., 1992; Poulton and Li, 1994). Four isoforms of AH, designated AH I, AH I′, AH II, and AH II′, were recognized. All are monomeric glycoproteins with similar kinetic properties but differing in their pI and N-terminal sequences. The sequencing of a near full-length AH cDNA that encodes AH I showed that this β-glucosidase belongs to glycoside hydrolase family 1 (Zheng and Poulton, 1995). Far less is known about PH, because no authentic cDNA clone has as yet been isolated from any species. However, in black cherry seeds, PH is a glycoprotein that exists as three isoforms, designated PH I, IIa, and IIb, that were separable by hydroxyapatite and Sephacryl S-200 chromatography (Kuroki and Poulton, 1987). Whether PH displays microheterogeneity in vegetative tissues was unknown until now, because its purification from such sources has not been reported.

To gain a better understanding of cyanogenesis in rosaceous stone fruits, we have now cloned and characterized a full-length cDNA (designated Ph1) that encodes seed isoform PH I. Using both biochemical and molecular approaches, we also investigated PH microheterogeneity in black cherry seedling shoots. Four novel PH cDNAs (named sequentially Ph2 through Ph5) were isolated and characterized, of which three were shown to encode specific PH isoforms purified here from the same source. Sequence analysis has indicated that the five PHs, like AH, belong to glycoside hydrolase family 1 and are probably encoded by a multigene family. Genomic sequences corresponding to the open reading frames (ORFs) of Ah1 and Ph1 through Ph5 were amplified from black cherry genomic DNA by long distance (LD)-PCR, allowing analysis of their exon-intron organization. Finally, contributing to efforts to engineer the substrate specificities of β-glucosidases for particular biotechnological purposes, we have used computer modeling, sequence comparison, and site-directed mutagenesis to predict and alter amino acid residues that may confer upon AH and PH their characteristic aglycone specificities.

Amino acids are given in the one-letter code, with unassigned residues indicated by X. Sequences have been aligned to maximize similarity.

Percentage amino acid identity was determined by the GAP program. GenBank accession nos.: {"type":"entrez-nucleotide","attrs":{"text":"U39228","term_id":"1155254"}}U39228, P. avium β-glucosidase (Wiersma and Fils-Lycaon, 1995); {"type":"entrez-nucleotide","attrs":{"text":"X56733","term_id":"21952"}}X56733, Trifolium repens linamarase (Barrett et al., 1995); and {"type":"entrez-nucleotide","attrs":{"text":"S35175","term_id":"249261"}}S35175, cassava (Manihot esculenta Crantz) linamarase (Hughes et al., 1992).

Activities are expressed in micromoles per milliliter enzyme per hour and are the average of three separate measures.

The following single, double, and triple mutations were introduced into the Ah1 cDNA by site-directed mutagenesis and verified by sequencing. The substrate specificities of the mutated recombinant AH1 toward amygdalin, prunasin, and PNPG were compared with that of unmutated, recombinant AH1. Activities are expressed in micromoles substrate hydrolysed per milliliter enzyme per hour and are the average of three separate measures.

ACKNOWLEDGMENTS

Footnotes

LITERATURE CITED