纤维素酶家族及其催化结构域分子改造的新进展

纤维素酶的分子改造是其催化性能改进及催化效率提升的重要手段。近年来,组学技术与结构测定技术的迅速发展,人们已建立了包括糖苷水解酶(Glycoside hydrolase,GH)在内的碳水化合物活性酶组分数据库。通过对同一蛋白家族进行序列比对、分子进化分析与祖先基因重构,以结构模建分析为指导的纤维素酶分子改造,可以明显缩小序列或结构的搜索空间,加快酶分子改造的速度,增大理性设计成功的概率;同时针对催化中心活性架构的分析可以进一步阐明纤维素酶的催化机理与酶分子持续性降解机制。文中主要对纤维素酶家族及其催化结构域的分子改造取得的最新进展作了综述。在后基因组时代基于蛋白质家族中的海量数据分析,以其保守结构信息为指导的理性设计,将会成为纤维素酶分子改造的重要方向,从而推动生物质转化工艺的快速发展。     

Phylogenetic analysis of family 6 glycoside hydrolases

Multiple sequence alignment separates members of glycoside hydrolase Family 6 into eight subfamilies: one of mainly actinobacterial endoglucanases (EGs), one of ascomycotal EGs, one of chytridiomycotal EGs and cellobiohydrolases (CBHs), one of actinobacterial and proteobacterial CBHs, one of chytridiomycotal CBHs, two of ascomycotal CBHs, and one of basidiomycotal CBHs. Each also has some proteins of unknown function. Multiple sequence alignment also extends to all of Family 6 the observation that lengths of loops that form the active-site tunnel in CBHs vary among subfamilies, and along with loop conformations, determine enzyme function.     

Comparison of glycoside hydrolase family 3 β-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the K_M values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X₂), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.     

Kinetic relationships with processivity in Serratia marcescens family 18 glycoside hydrolases

In nature, recalcitrant polysaccharides such as chitin and cellulose are degraded by glycoside hydrolases (GH) that act synergistically through different modes of action including attack from reducing-end and nonreducing-end (exo-mode) and random (endo-mode) on single polysaccharide chains. Both modes can be combined with a processive mechanism where the GH remain bound to the polysaccharide to perform multiple catalytic steps before dissociation into the solution. In this work, we have determined association rate constants and their activation paramaters for three co-evolved GHs from Serratia marcescens (SmChiA, SmChiB, and SmChiC) with an oligomeric substrate. Interestingly, we observe a positive correlation between the association rate constants and processive ability for the GHs. Previously, a positive correlation has been observed between substrate binding affinity and processive ability. SmChiA with highest processive ability of the three GHs bind with a k_on of 11.5 ± 0.2 μM⁻¹s⁻¹, which is five-fold and 130-fold faster than SmChiB (less processive) and SmChiC (nonprocessive), respectively.     

Automated docking to explore subsite binding by glycoside hydrolase family 6 cellobiohydrolases and endoglucanases

Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.     

Positive selection of three chitinase genes of the family 18 of glycoside hydrolases in mammals

The digestive enzyme chitinase degrades chitin, and is found in a wide range of organisms, from prokaryotes to eukaryotes. Although mammals cannot synthesize or assimilate chitin, several proteins of the glycoside hydrolase (GH) chitinase family GH18, including some with enzymatic activity, have recently been identified from mammalian genomes. Consequently, there is growing interest in molecular evolution of this family of proteins. Here we report on the use of maximum likelihood methods to test for evidence of positive selection in three genes of the chitinase family GH18, all of which are found in mammals. These focal genes are CHIA, CHIT1 and CHI3L1, which encode the chitinase proteins acidic mammalian chitinase, chitotriosidase and cartilage protein 39, respectively. The results of our analyses indicate that each of these genes has undergone independent selective pressure in their evolution. Additionally, we have found evidence of a signature of positive natural selection, with most sites identified as being subject to adaptive evolution located in the catalytic domain. Our results suggest that positive selection on these genes stems from their function in digestion and/or immunity.     

蜜环菌糖苷水解酶家族基因在菌索与菌丝间的差异表达分析

蜜环菌是一种兼性腐生和寄生的真菌,通过降解伴栽基质并为药用植物(天麻)或菌物(猪苓)提供营养物质,而糖苷水解酶是这一过程的主要酶类.本研究从蜜环菌Armillaria mellea 541菌株转录数据库中共获得糖苷水解酶家族基因170个,分布于39个亚家族.进一步分析发现,这些家族基因编码的糖苷水解酶家族蛋白(glyc...     

Analysis of functional divergence within two structurally related glycoside hydrolase families

Two glycoside hydrolase (GH) families were analyzed to detect the presence of functional divergence using the program DIVERGE. These two families, GH7 and GH16, each contain members related by amino acid sequence similarity, retaining hydrolytic mechanisms, and catalytic residue identity. GH7 and GH16 comprise GH Clan B, with a shared β‐jelly roll topology and mechanism. GH7 contains fungal cellobiohydrolases and endoglucanases and is divided into five main subfamilies, four of the former and one of the latter. Cluster comparisons between three of the cellobiohydrolase subfamilies and the endoglucanase subfamily identified specific amino acid residues that play a role in the functional divergence between the two enzyme types. GH16 contains subfamilies of bacterial agarases, xyloglucosyl transferases, 1,3‐β‐D ‐glucanases, lichenases, and other enzymes with various substrate specificities and product profiles. Four cluster comparisons between these four main subfamilies again have identified amino acid residues involved in functional divergence between the subfamilies. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 478–495, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Strategies to reduce end‐product inhibition in family 48 glycoside hydrolases

Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free‐energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity. Proteins 2016; 84:295–304. © 2016 Wiley Periodicals, Inc.     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。     

Study of the active site residues of a glycoside hydrolase family 8 xylanase

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.     

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.     

A remote but significant sequence homology between glycoside hydrolase clan GH-H and family GH31

Although both the alpha-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is, however, proposed for clan GH-H with GH31. A sequence alignment, based on the idea that residues equivalent in the primordial catalytic GH-H/GH31 (beta/alpha)(8)-barrel may not be found in the present-day GH-H and GH31 structures at strictly equivalent positions, shows remote sequence homologies covering beta3, beta4, beta7 and beta8 of the GH-H and GH31 (beta/alpha)(8)-barrels. Structure comparison of GH13 alpha-amylase and GH31 alpha-xylosidase guided alignment of GH-H and GH31 members for construction of evolutionary trees. The closest sequence relationship displayed by GH31 is to GH77 of clan GH-H.     

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.     

Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two β-strands.     

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml^?1 at productivities >130 U l^?1 h^?1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.     

Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase

1,4-beta-D-Xylan is the major component of plant cell-wall hemicelluloses. beta-D-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. beta-D-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any beta-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum beta-xylosidase comprises three distinct domains; a catalytic domain of the canonical (beta/alpha)(8)-barrel fold, a beta-sandwich domain, and a small alpha-helical domain. We have determined the structure in two forms: D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.     

Identification of Major Facilitator Transporters Involved in Cellulase Production during Lactose Culture of Trichoderma reesei PC-3-7

A compound showing antimicrobial activity was isolated from an oil-macerated garlic extract by silica gel column chromatography and preparative TLC. On basis of the results of NMR and MS analyses, it was identified as Z-4,5,9-trithiadeca-1,6-diene-9-oxide (Z-10-devinylajoene; Z-10-DA). Z-10-DA exhibited a broad spectrum of antimicrobial activity against such microorganisms as gram-positive and gram-negative bacteria and yeasts. The antimicrobial activity of Z-10-DA was comparable to that of Z-ajoene, but was superior to that of E-ajoene. Z-10-DA and Z-ajoene are different in respect of substitution of the allyl group by the methyl group flanking a sulfinyl group. This result suggests that substitution by the methyl group would also be effective for the inhibition of microbial growth.