纤维素酶纤维素吸附区的结构与功能

大多数纤维素酶含有催化区和可与纤维素结合且氨基酸序列较为保守的纤维素吸附区（ｃｅｌｌｕｌｏｓｅ ｂｉｎｄｉｎｇ ｄｏｍａｉｎ,ＣＢＤ）。纤维毒品及附区促进酶与底物的结合,有利于催化区以对溶性底物的作用,但对可溶性底物的催化作用无影响。对ＣＢＤ结构的研究和进一步的诱变研究揭示。纤维素吸附区是通过几个芳香族氨基酸结合到纤维素表面。有实验证明外切莆聚糖酶的ＣＢＤ对结晶纤维素有疏解作用。ＣＢＤ结构域已居功     

微紫青霉外切葡聚糖纤维二糖水解酶（CBHI）的纤维素结合结构域及其链结区非水解性破坏纤维素结晶区结构

天然纤维素的结晶区必需在内、外切纤维素酶的协同作用下,始可被降解,这是纤维素降解的限速步骤。内、外切纤维素酶均为β－1,4－糖苷键的水解酶,但单一的内、外切纤维素酶却都不能水解天然纤维素的结晶区。内、外切纤维素酶怎样协同降解纤维素的机理一直未得阐明,是天然纤维素降解机制研究中的难点。纤维素酶分子是由具有催化功能的催化结构域（catalytic domain,CD）和具有结合纤维素功能的纤维素结合（吸附）结构域（cellulse biding domain,CBD）及涟结它们的链结区（linker）序列组成。已知一细菌的CBD在吸附纤维素后,纤维素聚合物断裂形成短小纤维,但这一现象还未在真菌中有类似发现,通过对插入质粒pUC-18上的微紫青霉外切葡聚糖纤维二糖水解酶CBHI的 cDNA基因,进行系列序列定向缺失等体外操作,得到了催化结构域序列缺失的重组质粒,转化大肠杆菌JM109后,利用纤维素结合结构域CBD可吸附纤维素的特性,筛选到含CBD编码区的转化子PUC18G,生产出了LacZ－CBD融合蛋白,经木瓜蛋白酶有限酶切后,分离纯化得到了CBD结构域及其链结区称为：CBDCBHI。经X光衍射、红外光谱分析、热活力测定和扫描电镜观察表明,CBDCBHI吸附纤维素后,能够导致纤维素聚合物氢键断裂,结晶度减低和形成短纤维,从而在底物可及性上为内切葡聚糖酶的水解糖化作用提供了条件,为真菌内、外切纤维素酶协同降解天然纤维素的作用机制提供了实验支持,并提出了内切纤维素酶的水解作用可为外切纤维素酶吸附纤维素提供能量的推论。     

海栖热袍菌内切葡聚糖酶Cel12B与木聚糖酶XynA CBD结构域融合基因的构建、表达及融合酶性质分析

海栖热袍菌内切葡聚糖酶Cel12B是极耐热胞外酶,氨基酸序列分析表明不含有纤维素结合结构域(CBD),对结晶纤维素无活性,但同样菌种来源的木聚糖酶XynA有催化结构域和纤维素结合结构城。用同样极耐热酶CBD区域和Cel12B融合构建重组质粒pET-20b-Cel12B-CBD,经诱导表达后,对结晶纤维素有活性,酶学特性研究表明:最适反应温度为100℃、最适pH为5.8、在pH4.5~7.0时酶活力稳定,90℃保温2h仍有87%的酶活。     

人类线粒体肌酸激酶uMtCK的底物结合位点分析

研究人类线粒体肌酸激酶u Mt CK的结合位点,将其与底物肌酸和ATP结合有关的关键氨基酸进行突变,并对突变体进行酶动力学和圆二色谱数据分析,探讨这些关键氨基酸在底物识别和催化过程中的作用。结果显示,与野生酶相比,突变体Q313A和R336A的K_m~(Cr)分别提高了2.6和2.9倍,k_(cat)下降了19%和55%;同样地,与ATP结合相关的突变体R125A和R287A分别使得K_m~(ATP)升高了3.2和4.2,k_(cat)下降了72%和38%。以上结果表明突变体R125A、R287A、Q313A和R336A影响对底物的结合,同时也降低了酶促反应的速度。利用圆二色谱比较野生酶与不同突变体的二级结构并无明显变化,但进一步的结构模拟表明底物结合位点氨基酸在与底物之间的氢键对底物的识别和酶催化过程中发挥着重要作用。     

抗真菌药物作用靶酶羊毛甾醇14α去甲基化酶研究

羊毛甾醇14α去甲基化酶是普遍存在于高等植物、真菌和哺乳动物体内的P450蛋白,是氮唑类抗真菌药物作用靶酶.到目前为止已分别确定了高等植物、真菌和哺乳动物体内该酶的氨基酸序列.该酶对底物的催化包括三个单加氧步骤,涉及自由基的生成和消除,血红素辅基在酶催化过程中起重要作用.底物羊毛甾醇只能结合在酶活性位点血红素辅基Nc吡咯环上方,其余血红素吡咯环被氨基酸残基封闭.底物羊毛甾醇的3β羟基、Δ⁸⁽⁹⁾双键和17位侧链是与酶活性位点正确结合的关键官能团.该酶两大类抑制剂(底物类似物和氮唑类抗真菌药物)结构-活性关系研究可为进一步优化和设计新型高效酶抑制剂提供基础.     

O-糖基化起始糖基转移酶的活性与结构研究

多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T） 是催化N-乙酰氨基半乳糖（GalNAc）结合到蛋白质Ser或Thr上的糖基转移酶,是黏蛋白型O-糖基化修饰的起始糖基转移酶。ppGalNAc-T是一个酶家族,表达产物均为Ⅱ型膜蛋白。虽然氨基酸序列高度同源,但各成员具有独特的底物特异性和动力学特征。因此,ppGalNAc-T的底物作用机制是O-糖基化研究领域中的关键课题。近年来,通过利用定点突变及晶体结构解析技术,ppGalNAc-T中与底物相互作用的重要氨基酸残基以及由这些残基所形成的对底物结合起关键作用的空间构象逐渐被揭示,为了解ppGalNAc-T酶家族的底物作用机制及其蛋白结构与催化活性间的关系提供了理论依据。     

鉴别杰多霉素生物合成后修饰氧化酶JadH中参与底物结合或催化的关键残基

JadH是羟化脱水双功能酶,参与杰多霉素生物合成中的聚酮后修饰反应,将2,3-dehydro-UWM6催化为dehydrorabelomycin。为了分析杰多霉素生物合成途径中后修饰氧化酶JadH结合、催化底物的关键氨基酸,构建了JadH与底物复合物的三维结构模型。利用该模型并结合JadH同源蛋白氨基酸序列比对分析,推测出JadH活性中心中可能参与底物结合或催化的关键氨基酸(R50、G51、L52、G53、F100、R221、I223、P295和G298)。通过定点突变及体外酶学实验对这些位点的突变体的催化活性进行评价,结果显示这些突变株活性均显著低于野生型,表明这9个氨基酸是JadH参与底物结合或催化的关键氨基酸。     

荧光光谱法定量测定纤维素酶活性架构中单个氨基酸突变对底物结合力的影响

纤维素酶活性架构是酶分子中多个氨基酸残基构成的可结合并催化底物的功能区,其中色氨酸等芳香族残基在该区域中起着重要作用.本研究利用荧光光谱法,定量分析了纤维素酶Ch Cel5A活性架构中色氨酸与底物的结合动力学过程,通过色氨酸荧光猝灭的定量分析,确定了色氨酸特异性结合时的底物浓度范围,并且测定了Ch Cel5A活性架构中单个氨基酸突变导致的底物结合常数的变化,与催化动力学参数比较发现,荧光光谱法可准确表征纤维素酶与底物的结合力及其单个残基突变引起动力学参数的变化.此外,由于p NP中含有强的吸电子基团,因而以p NPC等为配体时会高估与色氨酸的结合常数约20~100倍.荧光光谱法可以测定纤维素酶结合糖分子底物的动力学参数,该方法具有灵敏和快速的特点,这为蛋白质与底物之间相互作用的定量分析提供了新的视角.     

持续性内切纤维素酶高效催化的研究进展

持续性内切纤维素酶(Processive endoglucanase)是一类新发现的双功能纤维素水解酶,既符合内切酶的作用特征,又具有外切酶的持续催化能力,可高效降解纤维素生成小分子寡糖。这类酶通常具有模块化结构,碳水化合物结合模块(CBM)对酶的持续催化活性及底物结合能力表现出不同的影响。综述了该领域相关研究的最新进展,分析了持续性内切酶潜在的研究方向及工业化应用的前景。     

长野芽孢杆菌普鲁兰酶的同源建模及三维结构分析

普鲁兰酶(Pullulanase)是脱支酶,因其能水解葡聚糖的α-1,6-糖苷键而有不同的工业应用潜力。本研究通过同源建模和分子对接的方法对长野芽孢杆菌(Bacillus naganoensis)普鲁兰酶进行建模及其三维结构分析,表明该酶由CBM41-X45aX25-X45b-CBM48-GH13_14多结构域组成,酶蛋白中心形成其催化区,催化区的Asp619、Glu648和Asp733三个残基构成酶的催化三联体。同时,通过柔性对接研究了酶与底物分子相互作用的关系,并预测构成酶的活性中心相关氨基酸残基,为进一步改良酶的特性提供重要的理论依据。     

Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases

Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.     

Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.     

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.     

Cellulose binding domains of a Phytophthora cell wall protein are novel pathogen-associated molecular patterns

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.     

C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates

Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C-terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C-terminal 140 amino acids of Ply500 and the C-terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non-covalent binding to surface carbohydrate ligands occurred in a rapid, saturation-dependent manner. We measured 4 x 104 and 8 x 104 binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD-ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram-positive bacteria.     

Biological pretreatment of cellulose: enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases

In this study, cellulose-binding domains (CBDs) of cellulases from Trichoderma reesei were used in a pretreatment step and were found to effectively reduce the crystallinity of cellulose (both Avicel and fibrous cellulose). This, in turn, led to higher glucose concentrations (up to 25% increase) in subsequent hydrolysis of cellulose using a mixture of cellulases and without the need for any intermediate purification step. CBDs were shown to be active in a range of temperatures (up to 50°C), while cellulase hydrolytic activity was greatly reduced after incubation at 50°C. This was explained by retention of full binding capacity after incubation at 50°C for 15 h. Our findings suggest that CBDs may be a valuable tool in pretreating cellulose and eventually afford faster enzymatic conversion of cellulose to glucose, thus contributing to more affordable processes in the production of biofuels.     

Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides.

Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A. Meinke, C. Braun, N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, J. Bacteriol. 173:308-314, 1991). The catalytic domain of 608 amino acids is at the N terminus. The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes. The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families. The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids. Deletion of these 131 amino acids reduces but does not eliminate activity. The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence. The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T. Watanabe, K. Suzuki, K. Oyanagi, K. Ohnishi, and H. Tanaka, J. Biol. Chem. 265:15659-15665, 1990). The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs). It functions as a CBD when fused to a heterologous polypeptide. Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD. The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity.     

Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene from Thermoanaerobacterium polysaccharolyticum

The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Escherichia coli. The open reading frame of manA is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da. The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative -35 (TTCGC) and -10 (TAAAAT) promoter sequence. The ManA of T. polysaccharolyticum is a modular protein. Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3). The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD. The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities. At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of the Bacillus-Clostridium cluster. The ManA of T. polysaccharolyticum, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure.     

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.     

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.