【作者】 夏思思；

【导师】 祁超；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 非核糖体多肽(NRPs)代表了微生物中大部分具有医药、农业和生物化学研究价值的多肽类天然产物。氨基酸腺苷化结构域(A结构域)是非核糖体多肽合成过程中的关键酶类,它负责结合和激活一个特异性识别的氨基酸底物并掺入其到NRP生物组装线上。Salinispora作为第一个被报道和确证的固有海洋性放线菌属,一直以来备受瞩目,而S.arenicola CNS-205正是该属放线菌中的代表菌株之一,其基因组中蕴含丰富的“沉默”次级代谢产物基因簇。为发掘海洋放线菌S arenicola CNS-205中的相关NRPS沉默基因簇,本研究采用PCR技术,从基因组总DNA中扩增出两个假定的氨基酸腺苷化结构域基因——sare0357和sare0718,并对两个基因的特征和可能的功能进行了生物信息学分析。分别构建了sare0357基因的原核重组表达质粒(pET23a-sare0357、 pET28a-sare0357、pET42a-sare0357)及Sare0718基因的原核重组表达质粒(pET23a-sare0718、pET28a-sare0718、pET42a-sare0718和pGEX-2T-sare0718)。将以上原核重组表达质粒转化E. coli BL21(DE3),经IPTG诱导表达后,进行了目的蛋白的亲和层析纯化及Western blotting鉴定。结果显示：在E. coli BL21(DE3)中表达的Sare0357重组蛋白主要以包涵体形式存在；在E. coli BL21(DE3)中表达的Sare0718重组蛋白也主要以包涵体形式存在,但其GST融合蛋白则获得了较明显上清形式的表达。为了进一步鉴定Sare0718-GST融合蛋白的生物化学功能,本文采用Thomas J.McQuade等新近提出的非放射性高通量鉴定A结构域的比色测定法(即无机焦磷酸酶偶联法结合孔雀石绿-钼酸铵化学显色定磷法),以20种常见蛋白质合成氨基酸作测试底物,对其进行了底物筛选和酶动力学参数测定。实验结果表明：Sare0718的确具有氨基酸腺苷化功能,在体外可通过ATP的消耗特异性识别和激活L-丙氨酸,进一步测得的酶动力学参数为Km=0.1164±0.0159(mM),Vmax=3.1484±0.1278(μM/min),kcat=12.5936±0.5112(min-1)。本论文首次对海洋放线菌S. arenicola CNS-205的两个未知氨基酸腺苷化结构域基因sare0357和sare0718进行了分子克隆和蛋白表达。对可溶形式表达的Sare0718-GST融合蛋白进行了体外酶活性测定,从而鉴定了一个新的L-丙氨酸腺苷化结构域。该工作为下一步探讨S. areniocola CNS-205中包含sare0718基因的完整未知NRPS基因簇及其“沉默”代谢产物的结构功能阐明奠定了基础。同时,本文也丰富了海洋放线菌来源NRPS腺苷化结构域的底物特异性和密码子领域的研究,为NRP组合生物合成及体外酶系合成预备了一个可用的新组件。更多还原

【Abstract】 Nonribosomal peptides (NRPs) represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during NRP biosynthesis. Salinispora arenicola CNS-205is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters.In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, sareO357and sare0718annotated "putative amino acid adenylation domain", were amplified from the genomic DNA by PCR. Firstly, the general features and possible functions of the two genes were predicted by bioinformatics analysis. Subsequently, a series of prokaryotic expression plasmids were constructed, including pET23a-sare0357, pET2Sa-sare0357and pET42a-sare0357, as well as pE723a-sare0718, pET28a-sare0718, pET42a-sare0718and pGEX-2T-sare0718. E.coli BL21(DE3) competent cells were then transformed with the above recombinant plasmids, respectively. After induction with IPTG, His-tagged recombinant proteins of Sare0357and Sare0718were expressed in E. coli BL21(DE3) mainly as Inclusion body whereas the GST-tagged Sare0718fusion protein was largely in the supernatant. All the recombinant proteins were purified with Affinity chromatography and verified by Western blotting. At the end, enzyme assays were performed to explore the adenylation activity of Sare0718in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that, among the20proteinogenic amino acids, Sare0718could in vitro specially recognize and activate L-alanine under the consumption of ATP. The basic kinetic parameters of Sare0718for L-alanine are Km=0.1164±0.0159(mM), Vmax=3.1484±0.1278(μM/min), kcat=12.5936±0.5112(min-1).In summary, this dissertation reported the molecular cloning and protein expression of sare0357and sare0718. By revealing the biochemical role of sare0718gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic NRPS-related gene cluster covering sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on.更多还原