Beta-Glucosidase
Created by Steven Kern
β-Glucosidases are important proteins found in all domains of living organisms (1). These enzymes hydrolyze the β-glucosidic bonds between aglycone and glycone moieties (1) (2) (3). In plants, β-glucosidase hydrolysis is the mechanism for activation of secondary metabolites that exist in a non-toxic glucosylated form (1) (2). The bioactivation of the aglycone species via hydrolysis is a biocidal response to tissue damage of many plant species when being fed on by insects (4), making β-glucosidases crucial to the innate resistance of many species to pests and disease. Studying the way that structure affects function for these biochemical defense methods is extremely significant for plants, such as wheat, on which millions of people depend for sustenance.
Mutant β-glucosidase in wheat complexed with DIMBOA-Glc (3AIS) is the inactive variant of a specific protein that catalyzes benzoxazinoids in Triticum aestivum, commonly known as wheat (2). This inactive mutant is the protein of interest for this study, and is designated as TaGlu1b-E191A. The enzyme is composed of 1 subunit, has a molecular weight of 64029.71 Da, and has an isoelectric point of 5.39 (5).
DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and DIMBOA (2,4-dihydroxy-7 -methxoy-1,4-benzoxazin-3-one) are two benzoxazinoids that are used in plants as defense against insects, fungi, and bacteria (3) (4). Transgenic research has shown that bioactivation of these secondary metabolites by β-glucosidase in wheat may also slow the spread of infection (6). Both benzoxazinoids exist normally in their non-toxic, glucosylated forms DIBOA-Glc and DIMBOA-Glc (4). When the plant tissue is injured, β-Glucosidases bioactivate DIBOA-Glc or DIMBOA-Glc via hydrolysis, yielding the toxic aglucones. These species spontaneously decompose to yield biocides formic acid and benzoxazolinones (4).
It is commonly accepted in literature that β-Glucosidases specificity favor the hydrolysis of DIMBOA-Glc and DIBOA-Glc in wheat and rye, respectively (3) (4). Not surprisingly, DIMBOA-Glc is more abundant in wheat, and DIBOA-Glc is more abundandt in rye (3) (7). A recent study published by Sue et al. shows that rye β-glucosidase activity is approximately equal for DIBOA-Glc and DIMBOA-Glc. The authors demonstrate that wheat β-glucosidase activity for DIBOA-Glc is equal to that of rye, but the wheat β-glucosidase activity for DIMBOA-Glc is significantly larger than that of rye (2). The specific structural differences that cause this functional difference are discussed later.
Position-Specific-Iterated Basic Local Alignment Search Tool (PSI-BLAST) is a search program that finds returns proteins with homologous primary structure (i.e. amino acid sequence) to the query protein. Highly homologous results will have an assigned E value less than 0.05. A PSI-BLAST search was preformed for TaGlu1b-E191A ("3AIS") through the NCBI Blast Server (8) (9).
Wheat has three known wild-type isozymes of β-glucosidase: TaGlu1a, TaGlu1b, and TaGlu1c (4) (2), which contain 569, 569, and 570 residues, respectively (10). TaGlub-E191A contains 565 residues (8) (9). The three wild-type isozymes all exhibited extremely similar primary structure to TaGlu1b-E191A, each with a E value of 0.0. Because TaGlu1b-E191A is a highly similar mutant of the wild type protein, hexameric β-glucosidase in wheat (2GDA) is a useful comparison protein. By observing change in function and analyzing change in structure of the mutation compared to the wild type protein, the impact of structure on function can be readily determined.
β-Glucosidase in Secale cereale (3AIU), commonly known as rye, also has a homologous primary structure with an E value of 0.0. This will be a useful comparison protein, because both species use the same secondary metabolite defense system. While wheat and rye both specifically hydrolyze benzoxazinoids, there are distinct differences in the substrate-specificity of each protein (4) (2).
In addition to the above proteins, two other comparison proteins were identified through the PSI-BLAST results. Os4BGlu12 (3PTK) in Oryza sativa L.(rice) had an E value of 2E-168, and cyanogenic β-glucosidase (1CBG) in Trifolium repens L. (white clover) had an E value of 6E-145 (8) (9). Both of these proteins are useful comparisons because they utilize different secondary metabolites from wheat and rye. Os4BGlu12 in rice hydrolyzes β-linked oligosaccharides and pNP-glycosides with unique specificity properties (11). Cyanogenic β-glucosidases hydrolyze cyanogenic glucosides, yielding toxic HCN species that block cellular respiration by inactivating mitochondrial cytochrome oxidase (4). These comparison proteins have highly homologous structure, yet analogous function, preferring different metabolite substrates.
A final comparison protein obtained through the PSI-BLAST search method wasβ-glucosidase in maize (1HXJ), returning an E value of 0.0 (8) (9). This protein has been researched extensively and there is a wide availability of structural information in the literature.
The secondary structure of TaGlu1b-E191A has 36% helical character. There are a total of 23 helices encompassing 206 residues. Both alpha helices and 3/10 helices are present. TaGlu1b-E191A also has 15% beta sheet character. There are 22 strands encompassing 86 residues (12).
Tertiary scructure homologies can be determined by searching the Dali Server, which finds matches by comparing intermolecular distances. A returned Z score about 2 means that the two proteins have similar tertiary structure. The Z scores for the comparison proteins 2GDA, 3AIU, 3PTK, and 1CBG were 69.6, 68.9, 59.0, and 55.0, respectively (13).
The tertiary structure of the β-glucosidase family is fairly similar across most plant species. However, there is a high degree of variability in the regions involved in oligomerization (4) (10). Thus, there is great diversity in the quaternary structure among plant β-glucosidases. Wheat TaGlu1a and TaGlu1b require the formation of homo- and heterohexamers to actively function (4) (10).
The hexameric β-glucosidase polymer is analogous to three combined dimmers. The N-terminal region of TaGlu1b is located between adjacent dimmers, and four hydrogen bonds link the subunits. By replacing the 25 N-terminal residues of TaGlu1b with those of maize glucosidase, which forms a dimer, the resultant macromolecules failed to form hexamers. This demonstrates the crucial role that the N-terminal sequence plays in maintaining dimer-dimer association. Furthermore, the maize-wheat hybrid β-glucosidases did not exhibit enzyme activity towards DIMBOA-Glc (10). This example demonstrates that structure affects function, and loss of structure causes loss of function.
β-Glucosidases have twomajor catalytic residues. Glu-407 acts as a nucleophile and Glu-191 acts as an acid/base catalyst. It should be noted that the protein of interest is inactive due to the mutation that replaces Glu-191 with Ala-191. These catalytic glutamic acid residues are located on opposite sides of the β-glucosidic bond of the bound substrate. The first step of catalysis involves Glu-407 performing a nucleophilic attack on the anomeric carbon of the aglycone moiety. This results in the generation of a glucose-enzyme intermediate complex. The acid catalyst, Glu-191 then protonates the glucosidic oxygen, which facilitates the aglycone dissociation. During this process, a necessary water molecule is activated by the catalytic residue to be used as a nucleophile for the hydrolysis of the glucosidic bond, which allows for the release of glucose (4). The critical impact of this water molecule is exhibited by the behavior of the inhibitor 2,4-Dinitrophenyl-2-deoxy-2-fluoro- β-glucoside.
2,4-Dinitrophenyl-2-deoxy-2-fluoro- β-glucoside inhibits wheat β-glucosidase by forming an intermediate complex. A covalent bond forms between Glu-407 of the catalytic center and an α-glucosidic bond of 2,4-Dinitrophenyl-2-deoxy-2-fluoro- β-glucoside. Hydrogen bonds form between the hydroxyl groups at positions 3-, 4-, and 6- and O1 of 2,4-Dinitrophenyl-2-deoxy-2-fluoro- β-glucoside and the side chains of Gln-43, Tyr-334, Glu-462, and Trp-463. 2,4-Dinitrophenyl-2-deoxy-2-fluoro- β-glucoside acts by replacing two nucleophillic water molecules that otherwise would be involved in an enzyme-glucose intermediate (2).
In the region ofthe binding site, the primary structures of wheat and rye β-glucosidases only differ at residues 464 and 465. TaGlu1a contains Ser-464 and Leu-465, while rye β-glucosidase contains Gly-464 and Ser-465 (10) (2). To test the impact of the differing structure on function, Sue et al. determined that the rye β-glucosidase mutations of G464S and S465L (i.e. eliminating the structural difference of the binding pocket) caused an increase in enzymatic efficiency for DIMBOA-Glc and a decrease in enzymatic efficiency for DIBOA-Glc (10).
These experiments prove that differences at residues 464 and 465 have a profound impact on protein function (2). While the dissimilar residues do not directly interact with DIMBOA-Glc, they do alter the width of the binding pocket. The structural differences make the binding pocket of rye β-glucosidase wider than that of wheat. This allows for greater substrate accessability to the binding pocket for rye, resulting in less restrictive substrate specificity (2).
One major structural difference unique to wheat and rye β-glucosidases is the presence of Tyr-378 in the binding pocket.The bulky tyrosine side chain restricts substrate access to the binding pocket. The aromatic residue acts as a lid to maintain the substrate in a favorable position (10).
In the binding pocket of TaGlu1b-E191A, DIMBOA-Glc is stabilized by π—π interactions between its aromatic ring and the sidechain of Trp-379. DIMBOA-Glc is further stabilized by a hydrogen bond with Thr-194 (2).
The catalytic glutamate, Glu-191 (which is substituted as alanine in TaGlu1b-E191A), is at an appropriate distance from the oxygen of the glucosidic bond of the aglycone moiety. The aglycone moiety is further stabilized by the aromatic residues Phe-198, His-205, and Trp-378. These three aromatic residues cause the 7-methoxy group of DIMBOA-Glc to locate closer to the entrance of the binding pocket (2).
The case of β-glucosidase in maize presents a valuable comparison opportunity, because the structure has been highly studied. The first major difference is the formation of oligomers. As discussed above, β-glucosidase in wheat forms a functional hexamer, and when hexamerization is not achieved, enzymatic activity is lost (10). β-glucosidase in maize characteristically forms a homodimer in solution (14). This difference in functional organization is stems from differences in the primary structure of the N-terminal residue sequences that play a key role in oligomerization (10).
Comparisons of maize and wheat β-glucosidases also demonstrate that disulfide bridges between cysteines are highly conserved (10). Maize β-glucosidase contains a disulfide bridge between Cys-205 and Cys-211 (14). Similarly, wheat β-glucosidase contains a disulfide bridge between Cys-210 and Cys-216 (10). Sulfate ions derived from LiSO4, which is part of the crystallization buffer, were fixate by complexing with Ser-366 and Asp-271 of one subunit and Arg-434 of another. This demonstrates that, like β-glucosidase in maize, β-glucosidase in wheat has asymmetric pairings of subunits (10).
Maize β-glucosidase has a slot-like active site. The walls are formed by four extended loops. The first contains residues Arg-54 through Ser-71. The second contains residues Tyr-195 through Glu-221. The third contains Arg-335 through Met-376. The fourth contains Trp-452 through Val-471. These loops are characterized by high variability among the β-glucosidase family. Structural differences in these loops directly correlate with substrate specificity differences (14).
Maize β-glucosidase can be used for comparison of wheat β-glucosidase by analyzing several key functional residues in the β-glucosidase family. β-glucosidase in maize stabilizes the aglycone moiety through hydrogen bonds with Gln-33, His-137, Arg-185, and Glu-459 (14). In wheat β-glucosidase however, hydrogen bond between the aglycone form with Gln-43, Tyr-334, Glu-462, and Trp-463 (2).
As discussed previously, the catalytic pair in wheat β-glucosidase are Glu 407 and Glu-191 (2). In maize, the catalytic glutamines are Glu-186 and Glu-401. These catalytic residues are part of highly conserved TXNEX and ITENG motifs common to many β-glucosidases. The distance between the carbonyl carbons of the two residues in maize β-glucosidase is 4.98 Å (14).
TaGlu1b-E191A is the protein of interest, and is an inactive wheat β-glucosidase. Researchers have used site-directed mutagenesis to produce similar inactive mutants in maize: maize β-glucosidase mutants E186D, E186Q, and E401Q. Each of these mutants exhibits a dramatically lower rate of enzymatic activity compared to the wild type (14). These findings are consistent with the analysis of TaGlu1b-E191A (2).
Analyzing the interactions of the catalytic glutamines in maize β-glucosidase can elucidate similar properties in wheat β-glucosidase, because of the conserved TXNEX and ITENG motifs. In the microenvironment of maize β-glucosidase,Glu-186 is protonated. The hydrophobicity of Trp-138 and Thr-189, which are located close to Oε1 of Glu-186, contribute to the increased acidity for catalysis at the optimum pH range of 5.5-6.0. Furthermore, Oε2 of Glu-186 participates in a hydrogen bond with Nδ2 of Asn-326. Unlike Glu-186, Glu-401 is surrounded by polar residues. Oε2 of Glu-401forms a salt bridge with Arg-91. Oε1 of Glu-401 forms two hydrogen bonds with the hydroxyl group of Tyr-328 and a water molecule. This means thatGlu-401 is deprotonated, making it available for nucleophilic attack (14). These findings are consistent with the behavior and function of the catalytic glutamines Glu-407 and Glu-191 of wheat β-glucosidase. This also sheds light on the mode of inactivation in the protein of interest TaGlu1b-E191A due to loss of the catalytic proton donor Glu-191 (2).
It is important that scientists continue to pursue understanding of the complex behavior of biochemical defense pathways for plants. Crops such as wheat, corn, and rice are the responsible for feeding a majority of the world’s population. Every year, people depend on the reliability and survival of these crops. These plants have evolved and perfected these biochemical defenses for millions of years. It is more important now than ever, with growing populations and famine in countries across the world, to study and utilize the natural defenses used by crops.