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．     

丝状真菌溶解性多糖单加氧酶的研究进展

溶解性多糖单加氧酶(lytic polysaccharide monooxygenases, LPMOs)是近些年才发现的一种蛋白,能够催化多糖葡萄糖苷键的氧化裂解,显著促进丝状真菌纤维素酶系对木质纤维素的降解作用。本综述对溶解性多糖单加氧酶发现过程、晶体结构、反应机制、活性位点和区域选择性、与纤维素酶的协同作用等方面分别进行了阐述,并对溶解性多糖单加氧酶对于木质纤维素降解方面的应用进行了论述。     

CYP116B家族单加氧酶的发现、表征及分子改造研究进展

CYP116B家族单加氧酶属于细胞色素P450单加氧酶的第IV家族,能够催化包括羟化、硫醚氧化、O-脱烷基、N-脱烷基和环氧化等在内的多种类型反应,具有广阔的应用前景。近年来,多个CYP116B家族成员酶的发现、分子改造及底物谱拓展使人们对它的酶学性质有了更为深入的理解,为开发新型具有工业应用潜力的CYP116B家族成员酶提供了研究基础。本文中,笔者主要从CYP116B家族单加氧酶的发现、表征、分子改造及结构功能关系等方面综述CYP116B在生物催化领域的研究进展。     

P450酶系中高效电子传递链的构建及应用

细胞色素P450作为单加氧酶的主要成员,能够在多种化合物中引入氧分子,催化包括羟化在内的多种反应。在级联的氧化还原反应中,需特定的电子传递链将氧原子中的电子传递至P450单加氧酶的亚铁红素结构中,并最终催化底物氧化,而电子传递体系的低效性往往成为整个反应的限速步骤。本文在介绍P450单加氧酶电子传递链基本结构的基础上,着重阐述对于细胞色素P450酶系中电子传递链未知或者内源性电子传递效率较低的情况下,利用DNA重组技术构建高效的电子传递链从而提高P450单加氧酶的催化效率相关研究进展,主要从细菌及真核细胞线粒体电子传递链(ClassⅠ)及真核生物细胞色素C还原酶CPR(ClassⅡ),天然融合电子传递链及人工融合蛋白电子传递链的构建及其应用展开。     

来源卤化二型聚酮类生物合成基因簇的两种关键功能基因鉴定与表达

为催化卤化产物,挖掘具有生物活性的新卤化物提供新的方法途径,拟构建卤化酶原核表达载体催化天然卤化物的卤化过程。对链霉菌Streptomyces sp. FJS31-2基因组中一个II型PKS类产物zunyimycin生物合成基因簇进行系列分析,针对其中的后修饰酶卤化酶基因和单加氧酶基因进行原核表达纯化。通过分子生物学手段成功构建卤化酶基因(65 kD)和单加氧酶基因(37 kD)的原核表达载体,并对其进行鉴定。卤化酶和单加氧酶在催化化合物的卤化过程中,会产生疑似新卤化产物。     

假单胞菌单加氧酶基因的克隆表达及其在手性亚砜生物催化合成中的活性分析

单加氧酶催化的硫醚底物不对称氧化反应是手性亚砜类药物绿色合成的新途径之一,而寻求高催化效率和高底物耐受性的硫醚单加氧酶仍是其难点和瓶颈。本研究从前期获得的能高效合成手性亚砜化合物的菌株中筛选和克隆了2个单加氧酶基因,构建相应的表达载体并诱导表达重组蛋白,最后以苯甲硫醚为底物来初步探讨重组蛋白在生物催化合成苯甲亚砜中的活性。结果显示,成功构建了2个基因的表达载体并获得了大量的可溶性重组蛋白。而且,这2个重组蛋白均表现出了一定的生物催化活性,能将少量的苯甲硫醚底物转化为苯甲亚砜产物。本研究为将来进一步利用重组酶蛋白生物催化合成手性亚砜类化合物打下了一定的基础。     

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。     

外源电子给体对甲烷单加氧酶催化丙烯环氧化反应活性的影响

以丙烯氧化反应为指标研究了不同外源电子给体对甲烷细菌(Methylomonas sp．GYJ30)休止细胞催化活性的影响。结果表明甲烷、甲醇、甲醛和甲酸盐作为电子给体加入反应中,将甲烷单加氧酶催化丙烯环氧化反应活性分别提高5．3,12．7,10和12．4倍。以甲烷和甲醛作为外源电子给体时提高初始浓度对甲烷单加氧酶具有抑制作用;而以甲醇和甲酸盐作为电子给体时提高初始浓度对甲烷单加氧酶催化活性无明显抑制作用。研究了甲醇作为电子给体时它的代谢、环氧丙烷的积累以及催化反应活性与反应时间的关系     

茶树菇非特异性过氧化酶的研究进展

惰性碳氢键(C—H)的选择性单加氧反应是有机合成的主要挑战之一,目前主要采用化学法和酶法催化,化学催化法往往反应条件苛刻,而酶催化法相对温和。来自茶树菇(Agrocybe aegerita)的非特异性过氧化酶(AaeUPO)是一类以H₂O₂为共底物的单加氧酶,可催化脂肪族和芳香族化合物的羟基化、环氧化及脱烷基等反应,因其具有电子传递过程简单、催化活性高、底物谱广等优点,是催化单加氧反应的高效生物催化剂。近年来,AaeUPO在分子改造、底物谱扩展和级联催化系统的应用等领域取得了一定进展,显示出较好的工业应用潜力。本文综述了AaeUPO的晶体结构及其在蛋白质工程方面的研究进展,重点探讨了AaeUPO催化的反应类型、底物范围以及级联催化系统构建中所面临的问题和挑战。     

细菌源吡咯里西啶类生物碱的发现及生物合成研究进展

吡咯里西啶类生物碱(Pyrrolizidine Alkaloids,PAs)在高等植物中分布广泛,目前超过6000种植物产生了650余个PAs。源于细菌的PAs发现较少,其中ClazamycinA和ClazamycinB由Umezawa等在1979年报道。近年来在微生物基因组和合成生物学发展的驱动下,细菌源PAs的发现和生物合成的研究方兴未艾。截至目前,已发现12类(60余个)源于细菌的PAs,包括波米西亚胺、Azetidomonamides和Brabantamides,以及含有PAs结构单元的多烯大环内酰胺Ciromicins和Heronamides。对这些结构多样、活性优异的细菌源PAs的研究发现,多数PAs依赖于一对独特的非核糖体多肽合成酶(Non-Ribosomal Peptide Synthetases,NRPSs)/拜耳-维利格单加氧酶(Bayer-Villiger Monooxygenase)生物合成其吡咯双烷基本骨架;而含有β-氨基酸的多烯大环内酰胺中吡咯双烷的形成则可能通过一个高度非对映选择性的电子重排反应途径。微生物基因组挖掘揭示了细菌中有大量沉默的PAs生物合成基因簇,说明细菌PAs在细菌进化和其环境/宿主的适应性中起着重要作用。     

First enantiodivergent Baeyer-Villiger oxidation by recombinant whole-cells expressing two monooxygenases from Brevibacterium

Microbial Baeyer-Villiger oxidations of representative mesomeric ketones with recombinant Escherichia coli cells expressing two monooxygenases from Brevibacterium were investigated. The two enzymes displayed enantiodivergent biotransformations on an array of structurally diverse substrates, allowing access to some key lactone intermediates in natural compound synthesis.     

Mapping the substrate binding site of phenylacetone monooxygenase from Thermobifida fusca by mutational analysis

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.     

Recombinant whole-cell mediated baeyer-villiger oxidation of perhydropyran-type ketones

Recombinant Escherichia coli cells expressing eight Baeyer-Villiger monooxygenases of bacterial origin have been utilized to oxidize prochiral heterocyclic ketones containing a pyran ring system. Within the biotransformation, two stereogenic centers were introduced with high control of enantioselectivity. The chemoselectivity of the enzymatic reaction was found to be high in favor of the Baeyer-Villiger process when using substituted ketone precursors incorporating functional groups labile to oxidation. A significantly different behavior was observed for two groups of monooxygenases with respect to substrate acceptance, which is consistent with our previous classification into two enzyme clusters.     

Modular and scalable biocatalytic tools for practical safety, health and environmental improvements in the production of speciality chemicals

Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with micro-bubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.     

Modular and scalable biocatalytic tools for practical safety,health and environmental improvements in the production of speciality chemicals

Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with micro-bubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.     

Expanding the biocatalytic toolbox of flavoprotein monooxygenases from Rhodococcus jostii RHA1

With the aim to enlarge the set of available flavoprotein monooxygenases, we have cloned 8 unexplored genes from Rhodococcus jostii RHA1 that were predicted to encode class B flavoprotein monooxygenases. Each monooxygenase can be expressed as soluble protein and has been tested for conversion of sulfides and ketones. Not only enantioselective sulfoxidations, but also enantioselective Baeyer–Villiger oxidations could be performed with this set of monooxygenases. Interestingly, in contrast to known class B flavoprotein monooxygenases, all studied biocatalysts showed no nicotinamide coenzyme preference. This feature coincides with the fact that the respective sequences appear to form a discrete group of sequence related proteins, distinct from the known class B flavoprotein monooxygenases subclasses: the so-called flavin-containing monooxygenases (FMOs), N-hydroxylating monooxygenases (NMOs) and Type I Baeyer–Villiger monooxygenases (BVMOs). Taken together, these data reveal the existence of a new subclass of class B flavoprotein monooxygenases, which we coined as Type II FMOs, that can perform Baeyer–Villiger oxidations and accept both NADPH and NADH as coenzyme. The uncovered biocatalytic properties of the studied Type II FMOs make this newly recognized subclass of monooxygenases of potential interest for biocatalytic applications.     

Exploring the structural basis of substrate preferences in Baeyer-Villiger monooxygenases: insight from steroid monooxygenase

Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP(+)-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP(+) is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.     

Purification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei strain KUFI-6N

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.     

Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP(+) only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP(+) interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP(+)) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.     

Resolution of fused bicyclic ketones by a recombinant biocatalyst expressing the Baeyer-Villiger monooxygenase gene Rv3049c from Mycobacterium tuberculosis H37Rv

Recombinant Escherichia coli B834 (DE3) pDB5 expressing the Rv3049c gene encoding a Baeyer-Villiger monooxygenase from Mycobacterium tuberculosis H37Rv was used for regioselective oxidations of fused bicyclic ketones. This whole-cell system represents the first recombinant Baeyer-Villiger oxidation biocatalyst that effectively resolves the racemic starting materials in this series. Within biotransformations using this organism one substrate enantiomer remains in high optical purity, while the second enantiomer is oxidized to one type of regioisomeric lactone preferably.