芳香聚酮后修饰氧化酶

氧化酶在芳香聚酮生物合成后修饰中普遍存在并对终产物的结构产生关键影响。本文简要总结了芳香聚酮后修饰氧化酶中几类最常见的氧化酶的结构和功能,并以杰多霉素生物合成途径中的后修饰氧化酶为例,阐明这些氧化酶在后修饰反应中发生作用的方式。并对后修饰氧化酶在组合生物学中的应用做了展望。     

三烷基取代芳香聚酮gombapyrones生物合成基因簇的确认及其生物合成推导

【目的】本研究旨在确认链霉菌Streptomyces rubellomurinus ATCC 31215来源芳香聚酮化合物(gombapyrones, GOMs)的生物合成基因簇(biosynthetic gene cluster, BGC),并对其生物合成途径进行推导。【方法】对链霉菌S. rubellomurinus ATCC 31215进行大规模发酵及提取分离,得到GOM-B和GOM-D;以三烷基取代芳香聚酮生物合成途径保守存在的P450单氧化酶的蛋白序列作为探针,在GOMs产生菌S. rubellomurinus基因组中进行BLAST搜索获得潜在的GOMs生物合成基因簇(gom BGC);通过对gom BGC中的聚酮合成酶(polyketide synthase, PKS)结构基因进行同框缺失突变,对突变株发酵产物进行高效液相色谱-质谱(highperformanceliquidchromatography-massspectrometry,HPLC-MS)分析以确认gomBGC与GOMs的产生相关;基于生物信息学分析,推导GOM-B的生物合成途径。【结果】从S. rubell...     

放线菌模块型聚酮合酶的系统发育组学分析及其在聚酮类化合物筛选中的应用

【目的】通过分析模块型聚酮合酶(polyketide synthase,PKS)的系统进化关系,阐明酮基合成酶(ketosynthase,KS)和酰基转移酶(acyltransferase,AT)序列与聚酮产物之间的关系,为放线菌天然产物的筛选提供指导。【方法】从PKSDB数据库的20个模块型PKS基因簇中调取所有KS(190个)和AT(195个)氨基酸序列,利用MEGA 4.0软件分别构建KS、AT、KS+AT 3种序列模式的系统发育树,并计算KS序列的簇内和簇间平均进化距离。设计了一对KS结构域的引物,通过PCR方法对20株活性放线菌分离菌株进行了筛选,测定了阳性菌株的KS序列,和已知的相关KS序列构建系统发育树,并对阳性菌株进行了发酵培养和代谢产物分析。【结果】放线菌来源的同一PKS的KS序列倾向于聚成一个进化枝,且按照其产物结构聚类;同一PKS的KS簇内平均进化距离小于0.300,不同PKS的KS簇间平均进化距离一般大于0.300。AT系统发育树按照其底物特异性聚成两个大的分枝;同一PKS的部分AT分别处于两个分枝,其余AT散在分布。KS+AT系统发育树则综合了KS树和AT树的拓扑结构特点。获得13株KS阳性分离菌株,它们的多数KS序列按照菌株分别聚类,其中4株菌的大部分KS各自聚成独特的簇,5株菌的大部分KS分别处在已知PKS进化枝内。从3株阳性菌中分离到预期的聚酮类产物。【结论】放线菌中KS的进化方式以垂直进化为主,而AT则以水平进化为主;KS序列与产物结构相关,且KS簇间平均进化距离可作为不同PKS的判定标准;相对于AT和KS+AT,KS系统发育组学分析更适用于指导放线菌聚酮类产物的筛选。     

长松萝中一个聚酮合酶基因簇的克隆和鉴定

【目的】探索药用地衣长松萝(Usnea longissima Ach)聚酮化合物的生物合成基因簇,克隆聚酮合酶(PKS)基因并分析其功能。【方法】以长松萝地衣型真菌为材料,通过巢氏PCR获得聚酮合酶基因片段和原位杂交筛选基因组文库获得聚酮合酶基因及相邻基因簇。并对获得聚酮合酶进行分子系统进化分析和基因表达分析。【结果】获得药用地衣长松萝中的编码聚酮合酶基因UlPKS5的全长序列以及相邻修饰基因β-内酰胺酶和脱水酶。聚酮合酶UlPKS5含有酮体合成酶(KS),酰基转移酶(AT),产物模板(PT)以及酰基载体蛋白(ACP)结构域。分子系统进化分析显示UlPKS5属于非还原型聚酮合酶中第五组,与蒽醌类化合物生物合成相关。通过半定量RT-PCR分析表明山梨醇(10%)和蔗糖(2%和10%)能够强烈诱导UlPKS5基因表达。【结论】聚酮合酶(UlPKS5)及相邻修饰基因β-内酰胺酶和脱水酶与长松萝中蒽醌类化合物生物合成相关。     

植物Ⅲ型聚酮合酶的分子机制与应用前景

植物III型聚酮合酶能催化生成一系列结构各异、具有不同生理活性、包含查耳酮合酶基本骨架的植物次生代谢产物,这类次生代谢产物不仅使植物体本身的抗逆性提高,并且对人类健康医疗有很好的应用前景。以下综述了近年来从植物中克隆、鉴定III型聚酮合酶的研究进展,着重论述了其分子结构、催化反应的类型和机制、表达调控及其在转基因工程方面的研究和应用前景。这些研究将为有效地对其进行基因改造,合成一些难以化学合成的新型天然化合物奠定基础,并且为将来进一步开展III型聚酮合酶的转基因工程提供了参考。     

共价交联捕获聚酮合成中的酮基还原酶和酰基载体蛋白结构域的瞬时复合物

【背景】在模块化聚酮合成酶(polyketidesynthase,PKS)的催化过程中,催化结构域与同源酰基载体蛋白(acyl-carrierprotein,ACP)之间的蛋白质-蛋白质相互作用起重要作用,但这种瞬时可逆的相互作用难以捕捉分析。【目的】获得ACP和酮基还原酶(ketoreductase,KR)相互作用的蛋白复合物。【方法】在KR和ACP之间的Linker上插入烟草蚀纹病毒(tobacco etch virus,TEV)蛋白酶切位点,通过双功能马来酰亚胺试剂BMH将KR和ACP共价交联,随后TEV酶切检测交联结果。调整反应条件,使交联效率最大化。根据KR-ACP交联复合物与体系内其他蛋白标签和分子量的差异,通过亲和层析和凝胶过滤等纯化手段,获得纯度较高的KR-ACP稳定交联复合物。【结果】单独表达的KR和ACP结构域交联不成功,融合表达的KR+ACP双结构域可以有效交联,结合使用亲和层析和凝胶过滤等纯化手段成功获得纯度较高的复合物。该策略可运用于多个KR和ACP的共价交联。【结论】建立了捕获并纯化KR和ACP瞬时相互作用复合物的有效方法,为后期晶体结构的解析、KR与ACP相互作用机理的揭示及参与相互作用关键氨基酸的鉴定提供了实验基础。     

红色红曲菌M7中色素聚酮合酶基因敲除突变体的鉴定

摘要:【目的】研究红色红曲菌(Monascus ruber) M7中控制红曲色素合成的聚酮合酶基因(pksPT)的功能。【方法】对M7 中pksPT进行了生物信息学分析;借助农杆菌介导的红曲菌转化技术敲除M7中pksPT,获得pksPT缺失突变体(ΔpksPT),比较M7和ΔpksPT菌落形态、产孢能力、生长速度、色素和桔霉素产量的差异。【结果】pksPT全长8687 bp,编码蛋白含有2690个氨基酸,属于非还原Ⅲ型聚酮合酶,包括β-酮酯酰基合成酶(KS)、酰基载体蛋白(ACP)、酰基转移酶(AT)和甲基转移酶(ME)四种结构域,组合形式为KS-AT-ACPACP-ME。ΔpksPT的分析结果显示,pksPT的敲除不影响其产分生孢子和闭囊壳的能力;ΔpksPT不能产生任何一种红曲色素;其生长速度明显快于野生菌株M7;桔霉素产量较M7 提高了2.8倍。【结论】pksPT是M7中控制红曲色素合成的关键基因,红曲色素的合成显著影响红曲菌产桔霉素能力和生长速度。     

基于定点突变的植物Ⅲ型聚酮合酶结构与功能研究进展

植物Ⅲ型聚酮合酶(Polyketide synthases,PKSs)催化形成一系列结构迥异、生理活性不同的聚酮类化合物的基本骨架结构,是聚酮类化合物生物合成途径的关键酶。目前已从植物中克隆和鉴定了多种功能不同的Ⅲ型PKSs。定点突变技术是研究蛋白质结构与功能之间复杂关系的重要方法。文中综述了近年来基于定点突变的植物Ⅲ型PKSs结构与功能关系的研究进展,包括利用定点突变技术修饰各种可能影响植物Ⅲ型PKSs结构的氨基酸残基,来研究其对功能的影响(如控制起始底物的特异性、缩合反应次数以及中间产物环化方式),以期为植物Ⅲ型PKSs结构与功能关系的研究提供参考。     

拐芹倍半萜没药烷吉酮的结构修饰及其对H~+/K~+-ATP酶的抑制作用

三峡区域药用植物拐芹的根中富含倍半萜类抗溃疡活性成分没药烷吉酮,为可开发和利用的中草药资源。本文对该化合物进行了提取分离、结构修饰和初步的构效关系研究,从拐芹根茎中提取并分离了没药烷吉酮,通过选择性还原、缩合和加成反应制备了四个没药烷吉酮氨基甲酰腙衍生物。用核磁共振波谱、质谱、红外和元素分析等方法确证了其结构,并测试了其体外对H~+/K~+-ATP酶的抑制活性和细胞毒活性。没药烷吉酮还原衍生物2及4-氯苯基取代的氨基甲酰腙衍生物4d较阳性对照药物奥美拉唑具有更好的体外抗溃疡活性(IC5024μmol/L)。本文探明了没药烷吉酮衍生物的结构对体外H~+/K~+-ATP酶抑制活性的影响,为消化性溃疡的治疗提供了新型倍半萜类候选药物。     

链霉菌聚酮类次生代谢产物及其生物合成基因簇

摘要：聚酮类抗生素在工业、农业和医药方面都具有重要的商业价值,至今通过美国FDA认证的聚酮类药物超过40个。随着分子生物学的发展,人们开展了抗生素合成基因簇的研究,并以此为基础发展了组合生物学,形成天然产物化学与分子生物学相结合的跨学科研究领域。本文以链霉菌为对象,对链霉菌产生的聚酮类抗生素的药物应用、聚酮合成酶（PKS）的研究及聚酮类药物的开发策略与前景作了相关介绍。     

Structure and mechanism of chalcone synthase-like polyketide synthases

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure–function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 393–398. Received 14 June 2001/ Accepted in revised form 15 July 2001     

植物类型III聚酮合酶超家族晶体结构与功能

植物类型Ⅲ聚酮化合物合酶(PKS)催化合成多种植物次生代谢产物的基本分子骨架,参与植物体许多重要生物学功能的行使,一直是研究蛋白结构与功能关系、基于结构进行分子改造的重要模式分子家族。目前在蛋白质数据库(PDB)中有超过80个不同种属来源的类型Ⅲ PKS的三维结构被报道,其中包括了研究最为透彻的查尔酮合酶在内的7种酶的晶体结构,这些结构的发表对于阐明该类酶复杂多变的底物专一性、链延伸和不同的环化反应机制奠定了结构基础。三维空间结构解析以及基于定点突变的结构功能分析是进行酶工程、基因工程的基础。以下系统综述了植物类型Ⅲ PKS超家族晶体结构和功能的研究进展。     

Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases

The process by which α-stereocenters of polyketide intermediates are set by modular polyketide synthases (PKSs) when condensation is not immediately followed by reduction is mysterious. However, the reductase-incompetent ketoreductase (KR) from the third module of 6-deoxyerythronolide B synthase has been proposed to operate as a racemase, aiding in the epimerization process that reverses the orientation of the α-methyl group of the polyketide intermediate generated by the ketosynthase to the configuration observed in the 6-deoxyerythronolide B final product. To learn more about the epimerization process, the structure of the C2-type KR from the third module of the pikromycin synthase, analogous to the KR from the third module of 6-deoxyerythronolide B synthase, was determined to 1.88 Å resolution. This first structural analysis of this KR-type reveals differences from reductase-competent KRs such as that the site NADPH binds to reductase-competent KRs is occluded by side chains and the putative catalytic tyrosine possesses more degrees of freedom. The active-site geometry may enable C2-type KRs to align the thioester and β-keto groups of a polyketide intermediate to reduce the pK_a of the α-proton and accelerate its abstraction. Results from in vivo assays of engineered PKSs support that C2-type KRs cooperate with epimer-specific ketosynthases to set the configurations of substituent-bearing α-carbons.     

纤维二糖磷酸化酶和纤维寡糖磷酸化酶的研究与应用

自然界中一些厌氧的纤维素降解菌能够产生纤维二糖磷酸化酶(Cellobiose Phosphorylase,CBP)和纤维寡糖磷酸化酶(Cellodextrin Phosphorylase,CDP)磷酸化裂解纤维二糖和纤维寡糖.CBP和CDP属于糖苷水解酶94家族(Glycoside Hydrolase Family 9...     

螺原体的分类及其生物多样性研究进展

摘要：螺原体(spiroplasma)是基本形态为螺旋形,无细胞壁,具有滤过性,能独立生活的一类原核微生物,是研究生物的运动、代谢及性比生物作用机理的重要模式生物。自从1973年建立了螺原体属(Spiroplasma)以来,已从许多昆虫等节肢动物、植物中分离到大量的螺原体,根据血清学特性目前发现的螺原体被分为34个血清组(包括15个血清亚组),并已确立了37个螺原体种。目前螺原体的分类采用的是多相分类法,主要根据形态学、生理生化、血清学以及系统发育学特性进行分析。螺原体具有丰富的生物多样性,它们与宿主的相互关系包括共生、互生和致病三种。在我国开展螺原体的资源调查和生物多样性研究将有助于充分认识和利用这类重要的生物资源。     

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability

Peptidase E (PepE) is a nonclassical serine peptidase with a Ser-His-Glu catalytic triad. It is specific for dipeptides with an N-terminal aspartate residue (Asp-X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser-His-Asn “catalytic triad.” Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser-His-Asp “catalytic triad.” We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp-X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C-terminal tail which adopts a β-hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser-His-Asp catalytic triad exhibits Asp-X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp-X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.     

Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide biosynthetic pathway: insights into extender unit biosynthesis

Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506. Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules. The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4%. FkbI has a similar fold to acyl-CoA dehydrogenases. Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate. This structural observation coincided the genetic experiment done by Carroll et al. J. Am. Chem. Soc., 124 (2002) 4176. Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis. A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.     

Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2

Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a β-strand rich globular protein consisting of eight antiparallel β-strands and three N-terminal α-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family.     

GH57家族高温淀粉普鲁兰酶的结构与功能分析

摘要：淀粉普鲁兰酶(E.C. 3.2.1.1/41)同时具有淀粉酶(E.C. 3.2.1.1)和普鲁兰酶(E.C. 3.2.1.41)的功能,属于糖苷水解酶GH13和GH57家族。由于淀粉普鲁兰酶可以同时水解α-1,4和α-1,6糖苷键,在淀粉糖化工业中具有降低生产成本、提高生产效率和提高糖化率的作用。其中高温淀粉普鲁兰酶由于能够在淀粉工业液化条件下同时催化淀粉的液化和糖化反应,因此在淀粉糖化工业中更具有应用价值。另外,淀粉普鲁兰酶的双功能催化机制对酶学研究中也具有重要价值。该文就近年高温淀粉普鲁兰酶的结