组合蛋白质工程和代谢工程设计全细胞催化剂合成靛蓝和靛玉红

从嗜高温放线菌Thermobifida fusca中分离得到的苯基丙酮单加氧酶主要催化芳香族化合物的Baeyer-Villiger氧化反应。对该酶的结构和功能进行研究时,发现位于底物结合口袋的Met446位点突变可以赋予突变酶催化C-H键活化的新功能,氧化吲哚合成靛蓝和靛玉红,但产量仅为1.89 mg/L。为了获得合成靛蓝和靛玉红的全细胞催化剂,直接补加吲哚并不能提高细胞合成效率,补加吲哚的前体物质L-色氨酸可以使细胞合成靛蓝和靛玉红的能力提高4.5倍,达到8.43 mg/L。为了进一步提高细胞的生物合成效率,通过代谢工程改造大肠杆菌的糖代谢途径,阻断葡萄糖异构酶基因pgi,使磷酸戊糖途径代替糖酵解途径成为葡萄糖的主要代谢通路,从而为细胞提供更多氧化吲哚所需的辅因子NADPH,导致细胞合成靛蓝和靛玉红的效率进一步提高3倍,达到25 mg/L。通过组合蛋白质工程和代谢工程设计全细胞催化剂不仅可以高效地合成靛蓝和靛玉红,而且设计理念为相关全细胞催化剂的开发提供了一种新的策略。     

AA10家族裂解多糖单加氧酶的模块化组成及底物选择性的研究进展

AA10家族裂解多糖单加氧酶(lytic polysaccharide monooxygenases, LPMOs)主要分布于细菌中,因其具有催化纤维素和几丁质等结晶多糖氧化降解的特性,在工业生物质转化过程中具有极强的应用潜力,从而受到广泛关注。然而,AA10家族不同LPMOs作用的底物种类及氧化位点和氧化产物也不尽相同,LPMOs的结构与组成对其底物选择性的影响机制有待进一步探究。因此,本文综述了AA10家族LPMOs的模块化结构组成及其催化机制,梳理了AA10家族LPMOs的底物谱,系统总结了AA10家族LPMOs的结构、关键作用残基及多模块组合对底物选择性影响的最新进展,并展望了LPMOs在生物质转化和生物燃料工业中广阔的应用前景。     

来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶的手性识别过程研究

立体选择性是2-卤代酸脱卤酶最重要的性质之一,但目前其手性识别过程尚不明确,对其进行研究和解析具有重要意义。以来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶dehDIV-R为模型,研究了R-2-卤代酸脱卤酶的手性识别过程。首先通过测定反应产物的构型,确定dehDIV-R催化底物为S_N2反应。通过Discovery Studio 3.0对dehDIV-R进行同源模建及底物分子对接,由对接结果和序列比对确定dehDIV-R立体选择性的关键位点Asn236,预测dehDIV-R的立体选择性与反应时底物到达反应位置的空间位阻密切相关。对dehDIV-R进行虚拟突变,将Asn236位点突变成具有不同空间位阻的残基Ala和Ser,并分别与底物分子进行分子对接,预测突变酶的立体选择性。根据预测结果,对Asn236氨基酸残基进行定点突变,发现在Asn236突变为Ala后的A1酶显示出对RS底物的活力;在Asn236突变为Ser后的S1酶显示出与原始酶相反的立体选择性,实现了立体选择性的反转。与模型的预测结果相符,证明了模型的合理性。     

Bacillaene酮还原酶结构域的异源表达及底物特异性分析

Bacillaene生物合成过程中,聚酮合酶第一个延伸模块的酮还原酶结构域(Bac KR1)既催化α酮基的还原,也催化β酮基的还原,具有天然的底物宽泛性。为进一步研究该结构域的底物特异性,在大肠杆菌中对其进行了异源表达。体外酶学分析表明Bac KR1可以催化聚酮类底物(±)-2-甲基-3-氧代戊酸-乙酰半胱胺硫酯外消旋体的立体选择性还原,仅生成4种非对映异构体中的一种,此外Bac KR1还可以催化环己酮和对氯苯乙酮等非聚酮类底物的还原,暗示了聚酮合酶中酮还原酶结构域作为生物催化剂的潜力。     

生物分子机器——甲烷单加氧酶的研究进展

甲烷氧化菌中的甲烷单加氧酶能够在生理条件下选择性地以甲烷和氧气为底物生成甲醇,麻省理工学院的Lippard教授称它为"神奇的生物分子机器"。本文重点对生物分子机器甲烷单加氧酶的结构、编码基因及调控机制、催化反应机理等进行了综述,此外也简要介绍了甲烷单加氧酶的产生菌甲烷氧化菌的研究历史及分类。生物分子机器甲烷单加氧酶可催化甲烷氧化成甲醇,不仅为甲醇的生产提供了一种新颖的生产方法,而且对生物分子机器的设计也有借鉴意义。     

伯克霍尔德氏菌(Burkholderia sp.)2-萘酸单加氧酶基因(nmo)的克隆及表达

通过酶切连接将Burkholderia sp．JTl500的一段DNA片段(4．8kb)亚克隆到表达载体pUC18上,得到重组子pEKl23。测序后的pEKl23重组子4．8kb插入片段的序列已经登陆欧洲EMBL基因库,序列接受号为AJ566333。对这一DNA片段的序列分析显示,此DNA片段含有3个阅读框,且在这3个阅读框5’端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3,其阅读框长度为1158bp,编码386个氨基酸,与已报道的Ralstonia eutropha HF39羟化酶(单加氧酶,bec)氨基酸序列有64％的同源性。pEKl23对2-萘酸代谢途径中4个关键底物的转化实验结果显示,其基因产物仅对2-萘酸发生加氧转化反应,而且2-萘酸浓度有明显的降低,证实此基因是2-萘酸单加氧酶基因(nmo)。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中。SDS-PAGE结果表明,pXK3、pEKl23两重组子中2-萘酸单加氧酶表达量并没明显区别,但加氧酶酶活却存在显著的差别。推测在启动子后,单加氧酶阅读框前的两个阅读框的基因产物,对单加氧酶活有促进作用。     

伯克霍尔德氏菌（Burkholderia sp.）2-萘酸单加氧酶基因（nmo）的克隆及表达

通过酶切连接将Burkholderia sp. JT1500的一段DNA片段(4.8kb)亚克隆到表达载体pUC18上,得到重组子pEK123。测序后的pEK123重组子48kb插入片段的序列已经登陆欧洲EMBL基因库,序列接受号为AJ566333。对这一DNA片段的序列分析显示,此DNA片段含有3个阅读框,且在这3个阅读框5′端发现一启动子特异序列。再用酶切连接方法得到仅含一个阅读框的重组子pXK3,其阅读框长度为1158bp,编码386个氨基酸,与已报道的Ralstonia eutropha HF39羟化酶（单加氧酶,bec）氨基酸序列有64%的同源性。pEK123对2萘酸代谢途径中4个关键底物的转化实验结果显示,其基因产物仅对2萘酸发生加氧转化反应,而且2萘酸浓度有明显的降低,证实此基因是2萘酸单加氧酶基因（nmo）。同时发现其基因产物也可以转化苯甲酸钠。该酶对苯甲酸的加氧转化途径正在研究中。 SDSPAGE结果表明,pXK3、pEK123两重组子中2萘酸单加氧酶表达量并没明显区别,但加氧酶酶活却存在显著的差别。推测在启动子后,单加氧酶阅读框前的两个阅读框的基因产物,对单加氧酶活有促进作用。     

来源于西梅的(R)-醇腈酶促立体选择性转氰合成(R)-酮醇腈

在水/有机溶剂双相反应体系中,研究了来源于西梅的(R)-醇腈酶催化酮与丙酮醇腈合成(R)-酮醇腈的立体选择性转氰反应．系统探讨了不同酶源、酶粉颗粒大小、底物浓度、两底物配比、酶浓度和底物结构对转氰反应的影响．结果发现西梅醇腈酶能高效催化三甲基硅酮与丙酮醇腈的立体选择性转氰．酶粉颗粒大小以直径0.3～0.45 mm为优,底物浓度以21 mmol/L左右为佳,底物丙酮醇腈与三甲基硅酮摩尔浓度比以2∶1为宜,酶浓度以60.9 g/L左右为好．西梅醇腈酶对3, 3-二甲基-2-丁酮几乎没有催化活性,而对其硅结构类似物三甲基硅酮却具有非常高的立体选择性和催化活性,在上述优化反应条件下反应24 h的底物转化率和产物光学纯度均高达99%以上,表明底物中的硅原子对西梅醇腈酶的催化活性有非常显著的促进作用．     

甲烷单加氧酶的催化性能和活性中心结构

甲烷单加氧酶是甲烷利用细菌代谢甲烷过程中的重要酶系,它能够催化烷烃羟基化和烯烃环氧化反应;还能催化降解氯代烃类,可用于环境中氯代烃类化合物污染的治理,是具有广泛应用前景的生物催化剂．甲烷单加氧酶是含有μ－氧桥双核铁催化活性中心的蛋白,它的研究对分子氧的活化、化学催化剂的设计具有重要意义．文章介绍了甲烷单加氧酶催化性能和机理的最新研究进展．     

裂解性多糖单加氧酶及其应用研究进展北大核心

裂解多糖单加氧酶(lytic polysaccharide monooxygenases,LPMOs)是近几年新发现的氧化酶,该酶在生物质酶解方面发挥着重要的作用,因此,被描述为生物质解构助推器。LPMOs与底物的结合具有特异性,催化机理尚未完全阐明。虽然关于LPMOs的研究很多,但真正投入到工业生物质转化中的却很少,这对它们的表达、调控和应用都提出了挑战。本文首先系统综述了LPMOs的发现与分类、催化机制、构效关系,其次探讨了LPMOs的活性测定方法及重组表达技术,最后协同综述了LPMOs在不同领域的应用并对未来的研究方向进行了展望。本综述有助于加深对LPMOs的系统认识,推动LPMOs及其酶工程的研究,以期为LPMOs的研究和应用提供参考。     

甲烷单加氧酶的研究进展

甲烷氧化细菌在转化甲烷制造新型燃料、单细胞蛋白和新功能酶生产、污水处理等方面有着潜在的应用前景,因此,甲烷单加氧酶作为其代谢过程中重要的酶系也受到人们的广泛关注。我们简要综述了近年来对甲烷单加氧酶的性质、结构、催化机理等方面的研究,特别是对颗粒性甲皖单加氧酶的相关性质进行了详细的阐述。     

Hot spots for the protein engineering of Baeyer-Villiger monooxygenases

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts for the conversion of ketones to lactones or esters while also being able to efficiently oxidize sulfides to sulfoxides. However, there are limitations for the application of BVMOs in synthesis. In this review we provide an overview of the protein engineering studies aiming at optimizing different properties of BVMOs. We describe hot spots in the active sites of certain BVMOs that have been successfully targeted for changing the substrate scope, as well as the possibility to influence this property by allosteric effects. The identified hot spots in the active sites for controlling enantio- and regioselectivity are shown to be transferable to other BVMOs and we describe concepts to influence heteroatom oxidation, improve protein stability and change the cofactor dependency of BVMOs. Summarizing all these different studies enabled the identification of BVMO- or property-dependent as well as universal hot spots.     

Biocatalyst assessment of recombinant whole-cells expressing the Baeyer-Villiger monooxygenase from Xanthobacter sp. ZL5

An Escherichia coli-based expression system for the Baeyer-Villiger monooxygenase (BVMO) from Xanthobacter sp. ZL5 was screened for whole-cell-mediated biotransformations. Biooxidation studies included kinetic resolutions and regiodivergent conversions of structurally diverse cycloketones. An extended phylogenetic analysis of the BVMOs currently available as recombinant systems with experimentally determined Baeyer-Villigerase activity showed that the enzyme originating from Xanthobacter sp. ZL5 clusters together with the sequences of bacterial CHMO-type BVMOs. The regio- and enantiopreferences experimentally observed for this enzyme are clearly similar to the biocatalytic performance of cyclohexanone monooxygenase from Acinetobacter as prototype for this group of BVMOs and support our previously reported family grouping.     

Alteration of the stereo- and regioselectivity of alkene monooxygenase based on coupling protein interactions

Alkene monooxygenase from Xanthobacter autotrophicus Py2 (XAMO) catalyses the asymmetric epoxidation of a broad range of alkenes. As well as the electron transfer components (a NADH-oxidoreductase and a Rieske-type ferredoxin) and the terminal oxygenase containing the binuclear non-haem iron active site, it requires a small catalytic coupling/effector protein, AamD. The effect of changing AamD stoichiometry and substitution with effector protein homologues on the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation has been studied. At sub-optimal stoichiometries, there was a marked change in regioselectivity, but no significant change in epoxidation stereoselectivity. Recombinant coupling proteins from a number of phylogenetically related oxygenases were investigated for their ability to functionally replace AamD. Substitution of AamD with IsoD, the coupling protein from the closely related isoprene monooxygenase, changed the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation, although this was accompanied by a high level of uncoupling. This indicates the importance of coupling protein interaction in controlling the catalytic specificity. Sequence analysis suggests that interaction between Asn34 and Arg57 is important for complementation specificity of the coupling proteins, providing a candidate for site-directed mutagenesis studies.     

Location of the receptor-interaction site on CheB, the methylesterase response regulator of bacterial chemotaxis.

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors, specifically methylation and demethylation of glutamates catalyzed by methyltransferase CheR and methylesterase CheB. The methylesterase is a two-domain response regulator in which phosphorylation of the regulatory domain enhances activity of the catalytic domain. In Escherichia coli and Salmonella typhimurium, a crucial determinant of efficient methylation and demethylation is a specific pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from sites of enzymatic action. Each enzyme binds pentapeptide, but the site of binding has been located only for CheR. Here we locate the pentapeptide-binding site on CheB by assessing catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with pentapeptide-CheB interaction by a CheB segment. The results place the binding site near the hinge between regulatory and catalytic domains, in a segment spanning the carboxyl-terminal end of the regulatory domain and the beginning of the linker that stretches to the catalytic domain. This location is quite different from the catalytic domain location of the pentapeptide-binding site on CheR and is likely to reflect the rather different ways in which pentapeptide binding enhances enzymatic action for the methyltransferase and the methylesterase.     

Structure of the complex of active site metal-depleted horse liver alcohol dehydrogenase and NADH.

The complex between active site-specific metal-depleted horse liver alcohol dehydrogenase and NADH has been studied with X-ray crystallographic methods to 2.9 A resolution. The electron density maps revealed that only the catalytic zinc ions are removed, whereas the non-catalytic zinc sites ae fully occupied. A gross conformational change in the protein induced by co-enzyme binding takes place in this enzyme species despite the absence of the metal ion in the catalytic center. This circumstance is of great importance in the understanding and further analysis of the trigger mechanisms operating during the conformation transition in alcohol dehydrogenase, since the catalytic center is located at the hinge region for a domain rotation in the subunit, and the metal atom is essential for catalysis. The overall protein structure is the same as that of an NADH complex of the native zinc enzyme and the co-enzyme is bound in a similar manner. The local structural changes observed are restricted to the empty metal binding site.     

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.     

Importance of a flexible hinge near the motor domain in kinesin-driven motility.

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.     

The viable but non-culturable state in the human pathogen Vibrio vulnificus

Abstract Genes encoding paniculate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides ( pmoA and amoA ) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.