木聚糖酶分子结构研究进展

本文详细叙述了木聚糖酶分子的催化区域、纤维素结合区域、木聚糖结合区域、连接序列与重复序列的结构和功能方面的研究进展，并介绍了木聚糖酶分子中常见的活性位点氨基酸以及该酶的定点诱变和三级结构。     

Triple point mutation Asp10 -> His, Asn101 -> Asp, Argl48 -> Ser in T4 phage lysozyme leads to the molten globule

The triple amino acid replacement (Asp10 His, Asn101 Asp,Arg148 Ser) in T4 phage lysozyme was carried out by site-directedmutagenesis. At acid pH (2.7) the mutant is in a confonnationalstate with the properties of the molten globule: (i) the mutantprotein molecule is essentially compact; (ii) its CD spectrumin the near UV region is drastically reduced in intensity ascompared with the wild type protein spectrum; (iii) the CD spectrumin the far UV region indicates the presence of pronounced secondarystructure in the mutant; (iv) unlike the wild type protein themutant protein can bind the hydrophobic fluorescent probe, ANS.     

Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H_1-2P₃H₄P_5-6H_7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H_1-2P₃H₄P_5-6H_7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H_1-2P₃H₄P_5-6H_7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C.     

Enzymatic Properties and Mutational Studies of Chalcone Synthase from Physcomitrella patens

PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.     

FgaPT2酶催化合成C-4异戊烯基化吲哚二酮哌嗪和定向诱变增强收率

在二甲基烯丙基二磷酸存在下,通过FgaPT2的酶催化合成C-4异戊烯化吲哚二酮哌嗪,对合成的产物进行了抗肿瘤、抗细菌、抗真菌、抗氧化活性测试,对生物活性最高的产物,研究了通过定点诱变提高酶合成产率的可行性。结果表明,FgaPT2酶催化合成了7个C4-异戊烯化吲哚二酮哌嗪,FgaPT2对底物具有一定的选择性,C4-异戊二烯化显著提高吲哚二酮哌嗪的生物活性,尤其是异戊二烯化产物 6b。Arg244的定点诱变表明,52.6%的FgaPT2突变体,提高了6b的合成产率,动力学参数验证了6a与突变FgaPT2之间的相互作用,可以提高异戊二烯基的合成产率。     

纤维素酶的蛋白质工程

概述了纤维素酶的6个家族的蛋白质工程研究进展,主要包括用基因定点突变技术对典型纤维素酶家族的三维构象、催化残基的确认。     

Residue Asn277 Affects the Stability and Substrate Specificity of the SMG1 Lipase from Malassezia globosa

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3₁₀4 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.     

Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ‐Cadinene Synthase into Germacradien‐4‐ol Synthase

δ‐Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ‐cadinene and about 2 % germacradien‐4‐ol, which is also generated from FDP by the cyclase germacradien‐4‐ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site‐directed mutagenesis of δ‐cadinene synthase. If W279 in δ‐cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS‐W279A is a catalytically highly efficient germacradien‐4‐ol synthase (k_cat/K_M=1.4×10^?3 μm s^?1) that produces predominantly germacradien‐4‐ol in addition to 11 % δ‐cadinene. Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release.     

Fluorescent properties of the Escherichia coli D-xylose isomerase active site

The consequences of active site mutations of the Escherichiacoli D-xylose isomerase (E.C. 5.3.1.5 [EC] ) on substrate bindingwere examined by fluorescence spectroscopy. Site-directed mutagenesisof conserved tryptophan residues in the E.coli enzyme (Trp49and Trpl88) reveals that fluorescence quenching of these residuesoccurs during the binding of xylose by the wild-type enzyme.The fluorescent properties of additional active site substitutionsat His101 were also examined. Substitutions of His101 whichinactivate the enzyme were shown to have altered spectral characteristics,which preclude detection of substrate binding. In the case ofH101S, a mutant protein with measurable isomerizing activity,substrate binding with novel fluorescent properties was observed,possibly the bound pyranose form of xylose under steady-stateconditions.     

定点突变提高细胞色素P450 BM-3吲哚羟基化能力

为进一步提高细胞色素P450 BM-3(A74G/F87V/L188Q)对吲哚的羟基化能力,根据酶结构与功能的关系,以突变酶E435T为基础,在168位点引入D168L突变,获得了吲哚羟基化能力得到显著提高的突变酶D168L/E435T。突变酶对吲哚的K_m为1.72 mmol·L^-1(父本2.09 mmol·L^-1),转化数(k_cat)为28.15 min^-1(父本4.04 min^-1),表明D168L定点突变可以略微提高酶对底物的亲和力,但主要的效应是促进了底物的转化速率,这两个效应的综合表现是使酶的催化效率(k_cat K_m^-1)比父本酶提高了8.48倍。此外,产物中副产物靛玉红的比例也降低为1.2%(父本7.3%),这说明该突变酶催化吲哚的区域选择性上也更有利于靛蓝的生成。     

Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.     

Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity

Analysis of a crystal structure of alcohol dehydrogenase (Adh)from horse liver suggests that Trp54 in the homologous yeastalcohol dehydrogenase prevents the yeast enzyme from efficientlycatalysing the oxidation of long-chain primary alcohols withbranching at the 4 position (e.g. 4-methyl-1-pentanol, cinnamylalcohol). This residue has been altered to Leu by site-directedmutagenesis. The alteration yields an enzyme that serves asan effective catalyst for both longer straight-chain primaryalcohols and branched chain alcohols.     

Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis

The unusual chemical properties of active-site Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodo-spirillum rubrumhave suggested that this residue is required for catalysis.To test this postulate Lys-329 was replaced with glycine, serine,alanine, cysteine, arginine, glutamic acid or glutamine by site-directedmutagenesis. These single amino acid substitutions do not appearto induce major conformational changes because (i) intersubunitinteractions are unperturbed in that the purified mutant proteinsare stable dimers like the wild-type enzyme and (ii) intrasubunitfolding is normal in that the mutant proteins bind the competitiveinhibitor 6-phosphogluconate with an affinity similar to thatof wild-type enzyme. In contrast, all of the mutant proteinsare severely deficient in carboxylase activity (< 0.01% ofwild-type) and are unable to form the exchange-inert complex,characteristic of the wild-type enzyme, with the transitionstateanalogue carboxyarabinitol bisphosphate. These results underscorethe stringency of the requirement for a lysyl side-chain atposition 329 and imply that Lys-329 is involved in catalysis,perhaps stabilizing a transition state in the overall reactionpathway.