Functional studies on wild-type and mutant Escherichia coli spermidine synthase by HPLC

以高效能液相層析儀分析原生型及突變型大腸桿菌的亞精胺合成酶功能性

楊雅婷

指導教授 ： 黃海美

國立清華大學/生命科學院/生物科技研究所/碩士(2010年)

https://doi.org/10.6843/NTHU.2010.00658

摘要

腸桿菌屬格蘭氏陰性之兼性厭氧菌，通常生長在溫血生物的腸道系統。大腸桿菌菌株K12的基因組序列已被發表於1997年。
來自於大腸桿菌K12的speE gene被預測可轉譯為亞精胺合成酶 (spermidine synthase,大腸桿菌亞精胺合成酶)，它是與speD (可轉譯出S-腺甘甲硫氨酸脫羧酶)共同組成一個單一的操作組。在多胺類生合成中，亞精胺合成酶主要催化腐胺 (putrescine, PUT)及去羧基的S-腺甘甲硫氨酸 (decarboxylated S-adenosylmethionine, dcSAM)合成亞精胺(spermidine)。 它的蛋白質序列與哺類類，植物，以及其它菌種的亞精胺合成酶的序列一致性 (sequence identity) 具有30-36 %。 本研究中，我們將speE基因 (含864個鹼基對)建構在pQE30質體中，使其在大腸桿菌SG13009中過量表現帶有六個組胺酸(histidine)的EcSpdS重組蛋白。此過量表現的蛋白(33.8 kDa)藉由鎳螫合親和層析法(Ni-NTA affinity chromatography)純化得到產率為30 mg/L 菌液。大腸桿菌亞精胺合成酶的酵素活性是以高效能液相層析儀分析，酵素活性是決定於一個反應終止後，產物SPD的生成及受質PUT的消耗。此大腸桿菌亞精胺合成酶在pH 7-8及溫度在37-43℃時能夠具有最大的催化活性。它的動力學常數(Km)對於腐胺(PUT)及去羧基的S-腺甘甲硫氨酸(dcSAM)分別為122.1 μM 及109.0 mΜ。在Lineweavre-Burk作圖中,當增加第一受質dcSAM的濃度時，相對於第二個受質PUT，能夠得到一組平行線，反之亦然。此暗指大腸桿菌亞精胺合成酶的機制為乒乓機制(ping pong mechanism)。
不同於原生型大腸桿菌亞精胺合成酶，在原生型的gatekeeping loop區上具有單一個殘基突變的突變型大腸桿菌亞精胺合成酶(D158A，C159A，C159S，T160A，D161A，P162A，I163A，P165Q)，它們的酵素活性皆分別使用薄膜色層分析(TLC)及高效能色層分析儀(HPLC)分析。結果顯示，D158A及D161A突變型重組蛋白的催化活性完全喪失。C159A, T160A及P165Q突變型重組蛋白的催化效率(catalytic efficiency)也分別下降為77%, 61%及57%。
目前為止，在任何物種皆未被指出其內生型的亞精胺合成酶的含量。以西方轉漬法，利用重組蛋白抗血清可偵測到大腸桿菌的細胞萃取液約含有0.083% (1/1250)的內生型亞精胺合成酶。相對於，胃幽門螺旋桿菌(Helicobacter pylori)(具有不完整的gatekeeping loop區域)的細胞萃取液約含有0.031%，當以此菌的重組蛋白抗血清可測得。若以免疫沉澱法收集內生型亞精胺合成酶(大腸桿菌或胃幽門螺旋桿菌)只能得到1/3的內生型亞精胺合成酶。

關鍵字

亞精胺合成酶 ； 腐胺 ； 亞精胺 ； 大腸桿菌 ； 高效能液相層析儀

並列摘要

Escherichia coli (E. coli) is a Gram-negative, facultative anaerobic bacterium that is generally found in the intestine of warm-blooded organisms. Whole genome sequencing in E. coli strain K12 has been completed in 1997. The speE gene from E. coli strain K12, coded for spermidine synthase (EcSpdS), shares an operon with speD gene (coded for S-adenosyl-methionine decarboxylase). Spermidine synthase catalyzes the production of spermidine (SPD) from putrescine (PUT) and decarboxylated S-adenosylmethionine (dcSAM) in polyamine biosynthesis. The deduced amino acid sequence of the EcSpdS shares 30-36 % sequence identities with most spermidine synthases from mammalian cells, plants and bacteria. In this study, the speE gene (864 bases pair, coded for EcSpdS) was cloned into the pQE30 vector and over-expressed the 6x His-tagged recombinant EcSpdS in Escherichia coli strain SG13009. The over-expressed protein (33.8 kDa) was purified by Ni-NTA affinity chromatography at a yield of 30 mg/L of bacteria culture. Enzyme activities were determined through HPLC to detect whether product spermidine formed or reactant putrecine consumed after reaction. Optimal pH and temperature for the EcSpdS reaction were between pH 7 - 8 and 37 - 43℃. The apparent Km values for putrescine and dcSAM were 122.1 μM and 109.0 μM, respectively. Increasing concentrations of the first substrate, dcSAM, gave an array of parallel lines (in Lineweavre-Burk plot) for the second substrate, putrescine, kinetics and vice versa, suggesting a ping pong mechanism for EcSpdS reaction.
The enzyme activities from EcSpdS mutants (D158A, C159A, C159S, T160A, P161D, P162A, I163A, P165Q), one residue different from wild type EcSpdS in gate-keeping loop region for each, were determined by both TLC and HPLC. The results showed that a complete loss of enzyme activity occurred in D158A and D161A mutants. Mutant protein, C159A, T160A and P165Q reduced enzyme catalytic efficiency to 77%, 61% or 57%, respectively.
Up to present, the content of endogenous spermidine synthase was not reported in any SpdS species. Endogenous EcSpdS protein was estimated about 0.083% (1/1250) in total cell lysate after western blotting along with anti-EcSpdS serum. In contrast, less amount (0.031%) of endogenous spermidine synthase (incomplete gate keeping region in structure) in total cell lysate from Helicobacter pylori after western blotting along with anti-HpSpdS serum. After immunoprecipitation (IP), only one-third of endogenous EcSpdS or HpSpdS was recovered after the detection with western blotting.

並列關鍵字

spermidine synthase ； putrescine ； spermidine ； Escherichia coli ； HPLC

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