Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Substrate specificity of Aspergillus oryzae family 3 β-glucosidase
Introduction
Widely distributed in bacteria, fungi, plants, and animals, β-glucosidase (EC 3.2.1.21, BG) is vital for many biological processes, such as biomass conversion (cellulolysis) by microbes/insects, biogenesis of various functional molecules (e.g., terpenols, flavonoids, phytohormones) from glycoside precursors, cyanide-based biodefense (CN releasing from cyano-glucoside), and degradation of various potentially harmful metabolites (e.g., glycosylceramides) (for a recent review, see [1]).
In addition to its physiological functions, BG is of great interest as a versatile industrial biocatalyst. Being indispensable for an efficient cellulase system, BG's hydrolytic activity on cellodextrins (particularly cellobiose) not only enables the final step of the cellulolysis of lignocellulosics (to yield glucose), but also relieves the product-inhibition on cellobiohydrolase and endoglucanase (two major enzymes responsible for the fragmentation/degradation of cellulose). Both of the catalytic functions of BG are critical to various bio-treatment/biorefinery processes, such as bioethanol production [1], [2].
The ability of BG to activate glycosidic bonds renders the enzyme highly interesting as a promising biocatalyst for the synthesis of stereo/regio-specific glycosides or oligosaccharides, molecules potentially useful as functional materials, nutraceuticals, or pharmaceuticals because of their biosignaling, recognition, or antibiotic properties [1]. Opposite to its hydrolytic activity, BG may catalyze a glycosidic bond formation via either a thermodynamically controlled “reverse” hydrolysis or a kinetically controlled transglycosylation [1], [3]. The main advantage of using BG over glycosyltransferase in such applications is that the former does not require any involvement of expensive/unstable nucleotide sugars precursors.
Among various BGs, microbial BG attracts increasing attention for its industrial application potential. In general, a secreted microbial BG may be advantageous over an animal/plant counterpart because its production, by fermentation, could be large scale, reliable, inexpensive, free of infectious viruses or prions, and environment-friendly. Aspergillus niger BG has been commercialized.
Understanding the fundamental structure–function relationships of BG is key to elucidating its catalytic mechanism and developing/optimizing its potential/performance as an industrial biocatalyst. For instance, heterogeneous biomass feedstocks may require a BG to possess specificity for di/oligosaccharides other than cellobiose in a biorefinery process, and specified glycosidic linkages/components may require a BG to discriminate different sugar isomers. To address such questions, insights of the structure and specificity of BG's active site are needed.
Based on amino acid sequence, BGs are grouped into two glycoside hydrolase families, GH1 and GH3 [4] (see http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html for updates). The three-dimensional structure of a dozen GH1 BG (in either resting, complexed, or transition state) has been determined. Consequently detailed information has been obtained with regard to key catalytic steps (substrate binding/activating, inhibitor binding). There are two three-dimensional structures known for the whole GH3 family (containing ∼ 600 known sequences), and only the one obtained from barley β-d-glucan glucohydrolase [5], [6] is functionally similar to typical microbial GH3 BG, although the sequence homology is low.
Although the progress in determining the three-dimensional structure of a GH3 BG lags, valuable kinetic and structural information has been accumulated. It is accepted that like its GH1 counterpart, GH3 BG carries out its catalysis via a “retaining”, double-displacement mechanism, involving an oxocarbenium transition state [5], [7], [8], [9], [10]. The catalytically important Asp nucleophile has been identified (in the GH3-wide conserved Ser–Asp–Trp segment) via site-directed mutagenesis [8] as well as sequence homology analysis against barley β-d-glucan glucohydrolase. Probing of GH3 BG's active site has been made by different hexopyranoses, glucosides, and derivatives [11], [12], [13].
Recently, we explored A. oryzae GH3 BG as a key component for a viable, industrial enzymatic system to convert corn stover biomass into fuel ethanol [2], [14]. To further develop the BG as a versatile and robust biocatalyst, we carried out a comparative and systematic study to probe the BG's specificity. By selectively altering the stereo and compositional feature of its substrate, we attempt to identify the key determinants in the BG responsible for substrate/inhibitor binding.
Section snippets
Materials and equipments
Chemicals used as reagents and buffers were commercial products of at least reagent grade. Trinder glucose reagent, pyranoses, glucosides, and inhibitors (Fig. 1) were from Sigma-Aldrich. α-l-Idose (l-Ido) was from Omicron Biochemicals. The glucose oxidase-based glucose detection kit was obtained from Pointe Scientific.
A. oryzae GH3 BG was cloned and expressed as previously reported [14]. Fig. 2 shows the ClustalW-based sequence alignment between the BG and the barley glucan glucohydrolase. The
Purification and initial characterization of A. oryzae GH3 BG
According to SDS-PAGE, the chromatographic purification yielded a BG preparation of > 95% purity, with an apparent molecular mass of ∼ 120,000. The difference between the observed value and sequence-predicted value (91,568) was attributed to glycosylation, since the deglycosylation with PNGase F and Endo H both led to a reduction of the SDS-PAGE molecular mass value. Based on its sequence, the BG had an isoelectric point of 4.9.
With pNP-Glc, the purified BG showed an apparent pH optimum at pH 5,
Discussion
The general molecular and kinetic properties, including substrate specificity and inhibitor susceptibility, of the recombinant A. oryzae GH3 BG were similar to the GH3 β-glucosidases originated from Aspergillus and others organisms [3], [8], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [30], [31]. In contrast, its substrate specificity and inhibitor susceptibility were quite different from the GH1 β-glucosidases of A. oryzae and other organisms [3],
Acknowledgment
We thank Dr. Robert L. Starnes of Novozymes for critical reading.
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