克雷伯杆菌甘油脱氢酶和1,3-丙二醇氧化还原酶的动力学机制

本研究主要对克雷伯杆菌甘油转化1,3-丙二醇代谢途径中的2个关键酶甘油脱氢酶(GDH)、1,3-丙二醇氧化还原酶(PDOR)反应机制和动力学进行了研究。首先,通过初速度和产物抑制动力学研究确定了GDH、PDOR双底物酶促反应机制为有序BiBi机制,明确了由反应物消耗到产物生成之间的历程。其次,建立了GDH、PDOR双底物酶促反应动力学模型,由动力学模型可知,在偶合反应中,如果GDH和PDOR酶量相同,GDH氧化反应成为限速反应,而辅酶I将主要以氧化型NAD+形式存在。动力学信息为酶法合成1,3-丙二醇和代谢工程研究提供理论指导。     

来源于克雷伯氏菌的1,3-丙二醇氧化还原酶的同源建模

用Swiss—Model和Modeller对来源于Klebsiella pneumonide的1,3-丙二醇氧化还原酶（PDOR）进行三级结构建模,并对所得的6个目标模型进行评价和比较,从中选择最好的一个模型,预测了辅酶NADP＋和Fe2＋在PDOR结构空间的近似位置,并定位了与NADP^＋和Fe^2＋作用的相关残基。     

弗氏柠檬酸菌1,3-丙二醇合成的代谢工程研究

【目的】研究弗氏柠檬酸菌(Citrobacter freundii) 1,3-丙二醇合成的代谢过程。【方法】构建甘油脱氢酶基因GSR-lacZ、1,3-丙二醇氧化还原酶基因PDO-lacZ和甘油脱水酶基因GL-lacZ等报告基因。在此基础上,构建3个相应的转座子突变文库。【结果】筛选到6株突变子,其相应关键酶表达水平提高1?11倍,1,3-丙二醇产量提高幅度为3%?50%。对转座子插入位点分析显示,5株突变子插入位点均为β-内酰胺酶(CKO_02592)编码基因,1株突变子插入位点为二氢硫辛酰胺基转移酶(CKO_02433)编码基因。进一步分析发现,β-内酰胺酶基因突变显著提高甘油脱水酶和甘油脱氢酶的表达水平,而1,3-丙二醇氧化还原酶表达水平没有变化;二氢硫辛酰胺基转移酶基因突变显著提高1,3-丙二醇氧化还原酶表达水平,其他两种关键酶基因表达水平不变。【结论】β-内酰胺酶和二氢硫辛酰胺基转移酶基因能够分别影响1,3-丙二醇合成代谢途径关键酶的表达,为构建工程菌株打下基础。     

大肠杆菌中1,3-丙二醇氧化还原酶的表达与纯化

目的:在大肠杆菌中表达1,3-丙二醇氧化还原酶(PDOR),并对PDOR进行纯化.方法:从克雷伯氏肺炎杆菌(Klebsiella pneumoniae)基因组中,克隆PDOR基因dhaT.构建表达载体pDK-dhaT,在E.coli DH5α中利用IPTG诱导进行表达.细胞裂解液利用硫酸铵盐析、Sephadex G-200凝胶层析和DE23 Cellulose阴离子交换层析,进行酶蛋白分离提纯.结果:用SDS-PAGE分析表明胞内PDOR占可溶性蛋白的39.8%,酶活为14.5U/ml.纯化后酶液比酶活提高3.94倍,回收率为15.5%.结论:成功地构建了PDOR高效表达载体,并且得到了高纯度的PDOR.     

产1,3-丙二醇菌株的诱变和筛选

为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

产1,3-丙二醇新型重组大肠杆菌的构建

利用PCR技术从大肠杆菌(Escherichia coli )中扩增出1.16 kb的编码1,3-丙二醇氧化还原酶同工酶的基因yqhD,将其连接到表达载体pEtac,得到重组载体pEtac-yqhD,重组载体在大肠杆菌JM109中得到高效表达。SDS_PAGE分析显示融合表达产物的分子量均为43 kD,同核酸序列测定所推导的值相符。对含有yqh-D的基因工程菌进行表达研究表明：37 ℃,以1.0 mmol /L IPTG诱导4 h,1,3-丙二醇氧化还原酶同工酶的酶活力达到120 u/mg蛋白,而对照菌株的酶活力为0.5 u/mg蛋白。再将含甘油脱水酶基因dhaB和含1,3-丙二醇氧化还原酶同工酶基因yqhD的重组质粒共转化大肠杆菌JM109得到重组大肠杆菌JM109（pUCtac-dhaB, pEtac-yqhD）,该菌株在好氧条件下,以1.0mmol/L IPTG诱导可将50 g/L甘油转化为38.0 g/L 1,3-丙二醇。首次发现1,3-丙二醇氧化还原酶同工酶在好氧条件下表现出较高的活性。     

弗氏柠檬酸菌dhaT基因的克隆表达、序列分析、酶的纯化及特性研究

1,3-丙二醇（1,3-propanediol,1,3-PD）是一种重要的化工原料,越来越受到广泛的关注。以弗氏柠檬酸菌(Citrobacter freundii)基因组DNA为模板,通过PCR得到1,3-丙二醇氧化还原酶(1,3-propanediol dehydrogenase,PDOR) 的基因dhaT,序列显示与来源于C.freundii DSM 30040 (Genbank U09771)相应基因的相似性为78%。将此基因构建于表达载体pSE380,得到重组质粒pSE-dhaT。重组质粒转化到宿主菌E.coli JM109中进行了表达,重组酶通过镍柱及Sephacral S-300进行纯化,重组酶SDS-PAGE结果显示有非常明显的单一的42kDa特异性蛋白条带出现。以丙醛为底物测定重组酶还原反应的最适温度为37℃、最适pH为8.0,对丙醛的Km值为10.05mmol/L,最大反应速度Vmax为37.27umol/ min /mg;以1,3-PD为底物测定重组酶氧化反应的最适温度为25℃、最适pH为10.5,对1,3-PD的Km值为1.28mmol/L,最大反应速度Vmax为25.55umol/min/mg。重组酶的还原反应比活为49.50U/mg,氧化反应比活为79.72U/mg。该酶同样具有假定的结合Fe2+的G-X-X-H-X-X-A-H-X-X-G-X-X-X-X-X-P-H-G模体保守结构。此研究为工程菌高效生产1,3-PD奠定了基础。     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。     

产1,3-丙二醇基因工程菌发酵条件的研究

利用途径工程的方法,将来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶基因dhaB和1,3-丙二醇氧化还原酶基因dhaT构建成多顺反子重组质粒pSE-dhaB-dhaT并在大肠杆菌JM 109中进行表达,在大肠杆菌中构建一条新的产1,3-丙二醇代谢途径。研究表明,重组菌株JM 109/pSE-dhaB-dhaT在微好氧条件下,尝试用廉价的乳糖为诱导物、维生素B12为辅酶,可以将甘油转化为1,3-丙二醇,产量达15.34 g/L,甘油转化率为35.7%,对低成本生产1,3-丙二醇作了有益的探索。     

Identification of two novel arginine binding DNAs.

RNA tertiary structure is known to play critical roles in RNA-protein recognition and RNA function. To examine how DNA tertiary structure might relate to RNA structure, we performed in vitro selection experiments to identify single-stranded DNAs that specifically bind arginine, and compared the results with analogous experiments performed with RNA. In the case of RNA, a motif related to the arginine binding site in human immunodeficiency virus TAR RNA was commonly found, whereas in the case of DNA, two novel motifs and no TAR-like structures were found. One DNA motif, found in approximately 40% of the cloned sequences, forms of hairpin structure with a highly conserved 10 nucleotide loop, whereas the second motif is especially rich in G residues. Chemical interference and mutagenesis experiments identified nucleotides in both motifs that form specific arginine binding sites, and dimethylsulfate footprinting experiments identified single guanine residues in both that are protected from methylation in the presence of arginine, suggesting possible sites of arginine contact or conformational changes in the DNAs. Circular dichroism experiments indicated that both DNAs undergo conformational changes upon arginine binding and that the arginine guanidinium group alone is responsible for binding. A model for the G-rich motif is proposed in which mixed guanine and adenine quartets may form a novel DNA structure. Arginine binding DNAs and RNAs should provide useful model systems for studying nucleic acid tertiary structure.     

Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster

We have solved the high-resolution crystal structures of the Drosophila melanogaster alcohol-binding protein LUSH in complex with a series of short-chain n-alcohols. LUSH is the first known nonenzyme protein with a defined in vivo alcohol-binding function. The structure of LUSH reveals a set of molecular interactions that define a specific alcohol-binding site. A group of amino acids, Thr57, Ser52 and Thr48, form a network of concerted hydrogen bonds between the protein and the alcohol that provides a structural motif to increase alcohol-binding affinity at this site. This motif seems to be conserved in a number of mammalian ligand-gated ion channels that are directly implicated in the pharmacological effects of alcohol. Further, these sequences are found in regions of ion channels that are known to confer alcohol sensitivity. We suggest that the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins.     

Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity

The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of intracellular environments. Here we demonstrate that natural HHRzs require the presence of additional nonconserved sequence elements outside of the conserved catalytic core to enable intracellular activity. These elements may stabilize the HHRz in a catalytically active conformation via tertiary interactions. HHRzs stabilized by these interactions cleave efficiently at physiological Mg2+ concentrations and are functional in vivo. The proposed role of these tertiary interacting motifs is supported by mutational, functional, structural and molecular modeling analysis of natural HHRzs.     

The hydrophobic lock-and-key intersubunit motif of glutathione transferase A1-1: implications for catalysis, ligandin function and stability

A hydrophobic lock-and-key intersubunit motif involving a phenylalanine is a major structural feature conserved at the dimer interface of classes alpha, mu and pi glutathione transferases. In order to determine the contribution of this subunit interaction towards the function and stability of human class alpha GSTA1-1, the interaction was truncated by replacing the phenylalanine 'key' Phe-51 with serine. The F51S mutant protein is dimeric with a native-like core structure indicating that Phe-51 is not essential for dimerization. The mutation impacts on catalytic and ligandin function suggesting that tertiary structural changes have occurred at/near the active and non-substrate ligand-binding sites. The active site appears to be disrupted mainly at the glutathione-binding region that is adjacent to the lock-and-key intersubunit motif. The F51S mutant displays enhanced exposure of hydrophobic surface and ligandin function. The lock-and-key motif stabilizes the quaternary structure of hGSTA1-1 at the dimer interface and the protein concentration dependence of stability indicates that the dissociation and unfolding processes of the mutant protein remain closely coupled.     

The crystal structure of pyrimidine/thiamin biosynthesis precursor-like domain-containing protein CAE31940 from proteobacterium Bordetella bronchiseptica RB50, and evolutionary insight into the NMT1/THI5 family

We report a 2.0 Å structure of the CAE31940 protein, a proteobacterial NMT1/THI5-like domain-containing protein. We also discuss the primary and tertiary structure similarity with its homologs. The highly conserved FGGXMP motif was identified in CAE31940, which corresponds to the GCCCX motif located in the vicinity of the active center characteristic for THi5-like proteins found in yeast. This suggests that the FGGXMP motif may be a unique hallmark of proteobacterial NMT1/THI5-like proteins.     

Structural organization of a viral IRES depends on the integrity of the GNRA motif

Little is known about the tertiary structure of internal ribosome entry site (IRES) elements. The central domain of foot-and-mouth disease (FMDV) IRES, named 3 or I, contains a conserved GNRA motif, essential for IRES activity. We have combined functional analysis with RNA probing to define its structural organization. We have found that a UNCG motif does not functionally substitute the GNRA motif; moreover, binding of synthetic GNRA stem-loops to domain 3 was significantly reduced in RNAs bearing UCCG or GUAG substitutions. The apical region of domain 3 consists of a four-way junction where residues of the GNRA tetraloop are responsible for the organization of the adjacent stem-loops, as deduced from ribonucleases and dimethyl sulfate accessibility. A single A-to-G substitution in the fourth position of this motif led to a strong RNA reorganization, affecting several nucleotides away in the secondary structure of domain 3. The study of mutants bearing UNCG or GUAG tetraloops revealed lack of protection to chemical attack in native RNA at specific nucleotides relative to the parental GUAA, suggesting that the GNRA motif dictates the organization and stability of domain 3. This effect is likely mediated by the interaction with distant residues. Therefore, the GNRA motif plays a crucial role in the organization of IRES structure with important consequences on activity.     

Modeling RNA tertiary structure motifs by graph-grammars

A new approach, graph-grammars, to encode RNA tertiary structure patterns is introduced and exemplified with the classical sarcin-ricin motif. The sarcin-ricin motif is found in the stem of the crucial ribosomal loop E (also referred to as the sarcin-ricin loop), which is sensitive to the alpha-sarcin and ricin toxins. Here, we generate a graph-grammar for the sarcin-ricin motif and apply it to derive putative sequences that would fold in this motif. The biological relevance of the derived sequences is confirmed by a comparison with those found in known sarcin-ricin sites in an alignment of over 800 bacterial 23S ribosomal RNAs. The comparison raised alternative alignments in few sarcin-ricin sites, which were assessed using tertiary structure predictions and 3D modeling. The sarcin-ricin motif graph-grammar was built with indivisible nucleotide interaction cycles that were recently observed in structured RNAs. A comparison of the sequences and 3D structures of each cycle that constitute the sarcin-ricin motif gave us additional insights about RNA sequence-structure relationships. In particular, this analysis revealed the sequence space of an RNA motif depends on a structural context that goes beyond the single base pairing and base-stacking interactions.     

Comparative analysis of polyphenol oxidase from plant and fungal species

Polyphenol oxidase from plants and fungi is a metalloenzyme containing a type-3 copper center and is homologous to oxygen-carrying hemocyanin of molluscs. Molluscan hemocyanin consists of two domains, an N-terminal domain containing the copper center and a smaller C-terminal domain, connected by an alpha-helical linker. It is presumed that the same is true of polyphenol oxidase from plants and fungi although the structure of a polyphenol oxidase containing the C-terminal domain has not been determined. We show that a number of important structural features are conserved in the N-terminal domains of polyphenol oxidases from various plants and fungi, including a tyrosine motif which can be considered a landmark indicating the beginning of the linker region connecting the N- and C-terminal domains. Our sequence alignments and secondary structure predictions indicate that the C-terminal domains of polyphenol oxidases are likely to be similar in tertiary structure to that of hemocyanin. Detailed bioinformatics analyses of the linker regions predict that this section of the polypeptide chain is intrinsically disordered (lacking fixed tertiary structure) and contains a site of proteolytic processing as well as a potential phosphorylation site.     

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse,ancient protein super family using motif search and structural modeling

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1γ (EF1γ) contains an N-terminal domain related to class θ glutathione S-transferases (GST). GST-like proteins related to class θ comprise a large group including, in addition to typical GSTs and EF1γ, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and β-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (α, μ, and π). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1γ is likely to form a fold very similar to that in the known structures of class α, μ, and π GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1γ is enzymatically active and that to exhibit GST activity, EF1γ has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.