In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations. The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.

β-Glucosidases (3.2.1.21) are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. β-Glucosidases function in glycolipid and exogenous glycoside metabolism in animals, defense, cell wall lignification, cell wall β-glucan turnover, phytohormone activation, and release of aromatic compounds in plants, and biomass conversion in microorganisms. These functions lead to many agricultural and industrial applications. β-Glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, and GH30, based on their amino acid sequences, while other β-glucosidases remain to be classified. The GH1, GH5, and GH30 β-glucosidases fall in GH Clan A, which consists of proteins with (β/α)(8)-barrel structures. In contrast, the active site of GH3 enzymes comprises two domains, while GH9 enzymes have (α/α)(6) barrel structures. The mechanism by which GH1 enzymes recognize and hydrolyze substrates with different specificities remains an area of intense study.

The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize ␤-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-␤-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile ␤-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-Å resolution, respectively. The structural data from the complexes allowed us to visualize an intact substrate, free aglycone, or a competitive inhibitor in the slot-like active site of a ␤-glucosidase. These data show that the aglycone moiety of the substrate is sandwiched between W378 on one side and F198, F205, and F466 on the other. Thus, specific conformations of these four hydrophobic amino acids and the shape of the aglycone-binding site they form determine aglycone recognition and substrate specificity in Glu1. In addition to these four residues, A467 interacts with the 7-methoxy group of DIMBOA. All residues but W378 are variable among ␤-glucosidases that differ in substrate specificity, supporting the conclusion that these sites are the basis of aglycone recognition and binding (i.e., substrate specificity) in ␤-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize ␤-glucosidases with high affinity without being hydrolyzed.

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ABSITRACTThe prolamin of maize (Zea mays L.), zein, was extracted from endosperm meal with 60% (v/v) 2-propanol/1% (v/v) 2-mercaptoethanol either directly or subsequent to extraction with 90% (v/v) 2-propanol. The zein extracted with 90% 2-propaol was essentially made up of 20 to 24 kilodalton polypeptides (cf-zein) while that exltactble with 60% 2-propanol/1% 2-mercaptoethanol containd, in addition to a-zein, 17 to 18 kilodlton methionine-rich polypeptides and a 27 kilodalton prolinerich polypeptide. While zein was separated into three fractions by differential solubility in 90% 2-propanol and 30% 2-propanol/30 millimolar sodium acetate (pH 6) using two different fractionation protocols. (23) from maize endosperm meal. Extractability, solubility, amino acid and electrophoretic analyses showed that a-zein constituted 35% of total zein, and included two prominent bands with mol wt 22 and 24 kD, respectively, and had an amino acid and polypeptide composition similar to that of whole zein (20). As for ,-zein, it failed to enter polyacrylamide gel without reduction but, after reduction, entered the gel and displayed three predominant size components with mol wts of 24, 22, and 14 kD. It also contained more histidine, arginine, proline, and methionine than did a-zein which Paulis (20) attnbuted to the presence ofthe 14 kD component in (#-zein. Alcoholsoluble reduced glutelin, or zein-2, was separated into two subfractions, water-soluble and water-insoluble, by dialysis against water (8,18).The same fraction was also separated into five subfractions by ion-exchange chematography (2). Two of these subfractions, 4 and 5, and a protein isolated by Wilson et al. (25), reduced-soluble protein, are now known to be the same as the water-soluble alcohol-soluble reduced glutelin isolated by Paulis and Wall (18).A simple fractionation scheme to permit separation of zein into fractions of unique polypeptide composition by differential solubility has been a subject of continuous investigation for the author in the last 7 years or so. After countless, mostly unsuccessful, experiments and years of painstaking effort such a procedure has been developed. This procedure, which separates the maize prolamin, zein, into three fractions each with unique polypeptide composition, is described and discussed in this report. MATERUILS AND METHODSPreparation of Cornmeal. Sources of maize seeds used for zein isolation were inbreds K55 and W64A. Endosperm meal was prepared from kernels whose germ and pericarp had been removed after soaking in water for 30 to 60 min. Endosperms were ground first in a mill and then by pestle in a mortar to pass a 150 um sieve. The corn meals so prepared were stored in a freezer with or without prior defatting with hexanes.

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Maize (Zea mays L.) ,-glucosidase (ft-d-glucoside glucohydrolase, EC 3.2.1.21) was extracted from the coleoptiles of 5-to 6-day-old maize seedlings with 50 millimolar sodium acetate, pH 5.0. The pH of the extract was adjusted to 4.6, and most of the contaminating proteins were cryoprecipitated at 0°C for 24 hours. The pH 4.6 supematant from cryoprecipitation was further fractionated by chromatography on an Accell CM column using a 4.8 to 6.8 pH gradient of 50 millimolar sodium acetate, which yielded the enzyme in two homogeneous, chromatographically different fractions. Purified enzyme was characterized with respect to subunit molecular weight, isoelectric point, amino acid composition, NH2-terminal amino acid sequence, pH and temperature optima, thermostability, and activity and stability in the presence of selected reducing agents, metal ions, and alkylating agents. The purified enzyme has an estimated subunit molecular mass of 60 kilodaltons, isoelectric point at pH 5.2, and pH and temperature optima at 5.8 and 500C, respectively. The amino acid composition data indicate that the enzyme is rich in Glx and Asx, the sum of which approaches 25%. The sequence of the first 20 amino acids in the N-terminal region was H2N-Ser-AIa-Arg-ValGly-Ser-Gln-Asn-Gly-Val-Gln-Met-Leu-Ser-Pro-(Ser?)-Glu-llePro-Gin, and it shows no significant similarity to other proteins with known sequence. The enzyme is extremely stable at 0 to 40C up to 1 year but loses activity completely at and above 550C in 10 minutes. Likewise, the enzyme is stable in the presence of or after treatment with 500 millimolar 2-mercaptoethanol, and it is totally inactivated at 2000 millimolar 2-mercaptoethanol. Such metal ions as Hg2+ and Ag+ reversibly inhibit the enzyme at micromolar concentrations, and inhibition could be completely overcome by adding 2-mercaptoethanol at molar excess of the inhibitory metal ion. The alkylating agents iodoacetic acid and iodoacetamide irreversibly inactivate the enzyme and such inactivation is accelerated in the presence of urea.in any other plant (8,28). Some maize inbreds lack the enzyme and are thus thought to be homozygous for a null allele of this locus (28). The recent data (4) show that the null phenotype is not due to the lack of enzyme activity; the enzyme is not detected in zymograms because it occurs as large quaternary structures and does not enter the gel.Maize f.-glucosidase was isolated by Huber and Nevins (10) as a buffer-soluble fraction and shown not to be active in the hydrolysis of,3-glucans. Nagahashi and Baker (20) performed differential centrifugation and centrifugation time course studies on maize j-glucosidase activity and found that the enzyme migrated past the ER marker only after a long centrifugation time. Nagahashi et al. (19) reported that the association of,-glucosidase activity with the cell wall in maize roots was pH-dependent, about sixfold greater at alkaline (7.7) than at acidic (6.0) pH values. They concluded that the cell wall-bound activity was not associated with the wall it...