Maize Β-glucosidase Research Articles

Plant Defensive β-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore.

Two-component activated chemical defenses are a major part of many plants’ strategies to disrupt herbivory. The activation step is often the β-glucosidase-catalyzed removal of a glucose moiety from a pro-toxin, leading to an unstable and toxic aglycone. While some β-glucosidases have been well studied, several aspects of their roles in vivo, such as their precise sites of enzymatic activity during and after ingestion, and the importance of particular isoforms in plant defense are still not fully understood. Here, plant defensive β-glucosidases from maize, white mustard and almonds were shown to resist digestion by larvae of the generalist lepidopteran Spodoptera littoralis, and the majority of the ingested activities toward both general and plant pro-toxic substrates was recovered in the frass. Among other proteins potentially involved in defense, we identified specific plant β-glucosidases and a maize β-glucosidase aggregating factor in frass from plant-fed insects using proteomic methods. We therefore found that, while S. littoralis larvae efficiently degraded bulk food protein during digestion, β-glucosidases were among a small number of plant defensive proteins that resist insect digestive proteolysis. These enzymes remain intact in the gut lumen and frass and can therefore further catalyze the activation of plant defenses after ingestion, especially in pH-neutral regions of the digestive system. As most of the ingested enzymatic activity persists in the frass, and only particular β-glucosidases were detected via proteomic analyses, our data support the involvement of specific isoforms (maize ZmGlu1 and S. alba MA1 myrosinase) in defense in vivo.

An automated method to evaluate the enzyme kinetics of β-glucosidases.

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

Retargeting a maize β-glucosidase to the vacuole – Evidence from intact plants that zeatin-O-glucoside is stored in the vacuole

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

Evolution and expression analysis of the β-glucosidase (GLU) encoding gene subfamily in maize

The maize β-glucosidase (ZmGLU1) hydrolyzes cytokinin-conjugates for releasing active cytokinins and thus plays important roles in cytokinin regulatory processes. ZmGLU1 belongs to glycosyl hydrolases 1 (GH1) gene family with a large number of members, and the gene function of other homologs remains to be investigated. In this study, 47 Arabidopsis, 34 rice, 31 brachypodium, 28 sorghum and 26 maize GH1 protein sequences were collected and subsequently used to construct a phylogenetic tree by Neighbor-Joining method. ZmGLU1 together with its 7 paralogs and 4 sorghum homologs were assigned into a distinct group (named GLU subfamily) with far evolutionary distance to other GH1 members. None of the Arabidopsis, rice and brachypodium gene falling into this group indicated a recent evolutionary emergence of GLU subfamily in some Poaceae plants after the divergence of Poaceae species. Phylogeny and comparative genome analysis revealed that GLU subfamily members of maize and sorghum evolved from a common ancestor, and expanded independently in each species by several duplications after maize-sorghum split. Ka/Ks analysis showed that purifying selection played important roles in maintenance of similar functions among the maize GLU paralogs. In addition, the similar protein properties and cytokinin-dependent gene expressions further suggested the similar functions of ZmGLUs in cytokinin activation. However, the organ-dependent expression of ZmGLUs exhibited diverse patterns, which might contribute to their diverse roles in cytokinin homeostasis. Taken together, this work put new insights into the evolution and expression of ZmGLU genes, and provided the foundation for future functional investigations.

A 40-bp A/T-rich repressor element involved in organ-dependent transcriptional regulation of ZmGLU1

Maize β-glucosidase (ZmGLU1) hydrolyzes cytokinin conjugates to release free cytokinins, and regulation of its expression is essential to control cytokinin homeostasis during plant growth and development. Although the promoter of ZmGLU1 gene was found to confer its organ-dependent expression, the underlying mechanism remained to be uncovered. In this study, by promoter deletion assay, a 40-bp A/T-rich sequence in ZmGLU1 promoter region ranging from −798 to −758 (T40) was identified as mediating the repression of promoter activity in seeds and shoot but not in root. Additional fusion of T40 upstream of the constitutive cauliflower mosaic virus 35S promoter (35S) suppressed 35S-driven gene expression in shoot and seeds. Electrophoretic mobility shift assay further revealed a specific interaction between T40 and an unknown binding protein in seeds and shoot but not in root. The negative correlation between ZmGLU1 gene expression/promoter activity and the intensity of T40–protein interaction suggests that T40 acts as a repressor cis-element involved in ZmGLU1 organ-dependent expression. Furthermore, this result provides a new A/T-rich sequence exhibiting transcriptional repression in higher plant.

Focused directed evolution of β-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening

Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired β-glucosidase activity. The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize β-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of β-glucosidases for either fundamental studies or potential biotechnological applications.

Deletion of the N-terminal dirigent domain in maize β-glucosidase aggregating factor and its homolog sorghum lectin dramatically alters the sugar-specificities of their lectin domains

Maize beta-glucosidase aggregating factor (BGAF) and its homolog Sorghum Lectin (SL) are modular proteins consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin (JRL) domain. BGAF is a polyspecific lectin with a monosaccharide preference for galactose, whereas SL displays preference for GalNAc. Here, we report that deletion of the N-terminal dirigent domain in the above lectins dramatically changes their sugar-specificities. Deletions in the N-terminal region of the dirigent domain of BGAF abolished binding to galactose/lactose, but binding to mannose was unaffected. Glucose, which was a poor inhibitor of hemagglutinating activity of BGAF, displayed higher inhibitory effect on the hemagglutinating activity of deletion mutants. Deletion of the dirigent domain in SL abolished binding to GalNAc, but binding to mannose was not affected. Surprisingly, fructose, an extremely poor inhibitor (minimum inhibitory concentration (MIC) = 125 mM) of SL hemagglutinating activity, was found to be a very potent inhibitor (MIC = 1 mM) of hemagglutinating activity of its JRL domain. These results indicate that the dirigent domain in this class of modular lectins, at least in the case of maize BGAF and SL, influences sugar specificity.

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu 50–Val 145) and the C-terminal (Phe 466–Ala 512) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile 72–Thr 82) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser 1–Thr 29, together with C-terminal region Phe 466–Ala 512, affects the size of Glu1–BGAF complexes. The dissociation constants ( K d ) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile 72–Thr 82 on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val 72–Glu 82 region was replaced with the Ile 72–Thr 82 region of Glu1). C-11 binds to BGAF, indicating that the Ile 72–Thr 82 region is indeed a major interaction site on Glu1 involved in BGAF binding.

Lysine-81 and Threonine-82 on Maize β-Glucosidase Isozyme Glu1 Are the Key Amino Acids Involved in β-Glucosidase Aggregating Factor Binding

In certain maize genotypes (nulls), beta-glucosidase specifically interacts with a chimeric lectin called beta-glucosidase aggregating factor (BGAF), resulting in high molecular weight complexes. Previously, we showed that three regions (S1-T29, E50-N127, and F466-A512) on the maize beta-glucosidase isozyme Glu1 are involved in interaction and aggregation with BGAF. Recently, we found that the peptide span I72-T82 within E50-N127 is essential and sufficient for BGAF binding, whereas the S1-T29 and F466-A512 regions are required for formation of large complexes. To define the contribution of individual amino acids in the above three regions to BGAF binding, we constructed mutant beta-glucosidases based on sequence differences between maize beta-glucosidase and sorghum beta-glucosidase (dhurrinase 2, Dhr2), which does not bind BGAF. Binding was evaluated by gel-shift assay and affinity by frontal affinity chromatography (FAC). In the gel-shift assay, Glu1 mutants K81E and T82Y failed to bind BGAF, and their FAC profiles were essentially similar to that of Dhr2, indicating that these two amino acids within the I72-T82 region are important for BGAF binding. Substitution of N481 with E (as in Dhr2) lowered affinity for BGAF, whereas none of the mutations in the S1-T29 region showed any effect on BGAF binding. To further confirm the importance of K81 and T82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four amino acids (I72, N75, K81, and T82) that differ between Glu1 and Dhr2 in the peptide span I72-T82 are required to impart BGAF-binding ability to Dhr2.

A new, sensitive method for enzyme kinetic studies of scarce glucosides

The maize β-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin- O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates.We developed a glucose assay using a system comprising three enzymes β-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine–Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-β- d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.

Ectopic over-expression of the maize β-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

Insights into the functional architecture of the catalytic center of a maize β-glucosidase Zm-p60.1

Insights into the functional architecture of the catalytic center of a maize β-glucosidase Zm-p60.1

Insights into the Functional Architecture of the Catalytic Center of a Maize β-Glucosidase Zm-p60.1

The maize (Zea mays) beta-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-A resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K(m) of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k(cat) was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.

Identification of β-Glucosidase Aggregating Factor (BGAF) and Mapping of BGAF Binding Regions on Maize β-Glucosidase

In certain maize genotypes (nulls), beta-glucosidase does not enter the gel and therefore cannot be detected on zymograms. Such genotypes were initially thought to be homozygous for a null allele at the glu1 gene. We have shown that a beta-glucosidase aggregating factor (BGAF) is responsible for the null phenotype, and it specifically interacts with maize beta-glucosidases and forms large insoluble aggregates. To understand the mechanism of the beta-glucosidase-BGAF interaction, we constructed chimeric enzymes by domain swapping between the maize beta-glucosidase isozymes Glu1 and Gu2, to which BGAF binds, and the sorghum beta-glucosidase (dhurrinase) isozyme Dhr1, to which BGAF does not bind. The results of binding assays with 12 different chimeric enzymes showed that an N-terminal region (Glu(50)-Val(145)) and an extreme C-terminal region (Phe(466)-Ala(512)) together form the BGAF binding site on the enzyme surface. In addition, we purified BGAF, determined its N-terminal sequence, amplified the BGAF cDNA by reverse transcriptase-polymerase chain reaction, expressed it in Escherichia coli, and showed that it encodes a protein whose binding and immunological properties are identical to the native BGAF isolated from maize tissues. A data base search revealed that BGAF is a member of the jasmonite-induced protein family. Interestingly, the deduced BGAF sequence contained an octapeptide sequence (G(P/R)WGGSGG) repeated twice. Each of these repeat units is postulated to be involved in forming a site for binding to maize beta-glucosidases and thus provides a plausible explanation for the divalent function of BGAF predicted from binding assays.

Crystal structure of a monocotyledon (maize ZMGlu1) β-glucosidase and a model of its complex with p-nitrophenyl β-d-thioglucoside

The maize β-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Å resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl β-d-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes.

The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize beta-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-A 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 beta-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 beta-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 beta-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize beta-glucosidases with high affinity without being hydrolyzed.

Substrate specificity of maize β-glucosidase

Maize ( Zea mays L.) β-glucosidase (β-glu) is a homodimer of 60-kDa monomers and localized in the plastid. Numerous glycosides were tested as substrates. When possible, K m and V max values were determined. The major natural substrate in methanol extracts of young maize seedlings was the glucoside of 2,4-dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3(4 H)-one (DIMBOA-glc) and this substance was hydrolyzed to glucose and DIMBOA by the enzyme. 4-Methylumbelliferyl-β- d-glucoside was the best substrate ( K m = 0.14 mM) for which kinetic data were obtained. Several aryl glycosides ( K m = 0.39−6.32 mM) and n- octyl-β- d- glycopyranosides were also substrates. Various compounds were also tested as inhibitors. When inhibition was observed, K i and/or K i′ values were estimated. Monosaccharides were poor inhibitors. The best competitive inhibitors were d-gluconic acid lactone, dhurrin, and (DIMBOA) ( K i values < 1 mM). Other glucosides, aglycones, and aglycone analogues were found to inhibit the enzyme competitively. Tryptamine was a mixed inhibitor ( K i = 33.9 mM, K i′ = 15.7 mM). The enzyme has a broad substrate specificity and can cleave many glycosides with hydrophobic aglycones; the best inhibitors and substrates are those that resemble the natural substrate DIMBOA-glucoside.

Tissue specificity and genetic control of the β-Glu null phenotype in maize

Sporophytic maize β-glucosidase (β- d-glucoside glucohydrolase; EC 3.2.1.21) is a dimeric enzyme expressed in several seedling and mature plant tissues. The enzymes shows a high degree of polymorphism and null individuals are frequent. The expression pattern of the enzyme was analysed spectrophotometrically and by zymogram analysis in different tissues of several maize genotypes (normal, nulls, F 1s and backcrosses). It is shown that the null phenotype is tissue specific, since the so called ‘null’ genotypes have a normal glucosidase activity in all tissues tested except the coleoptile. Coleoptilar extracts from F 1s between a null and normal lines also lack stainable β-glucosidase activity. It is hypothesized that the null phenotype is due to a dominant regulatory element that effects the correct assembly of the enzyme monomers into an active dimeric molecule. Analysis of the backcross generations to the null and to the normal parent indicated that the regulatory locus is independent of the structural locus Glul. In one backcross a highly distorted segregation yielding a large excess of null phenotypes was observed. The genetic basis of this phenomenon remains unclear, although the presence of gametophytic factors or of paramutation as possible causes are excluded.