胃幽門螺旋桿菌是一種格蘭氏陰性微需氧菌,全世界有百分之50的人口感染胃幽門螺旋桿菌,此菌會寄生在人類的胃部而導致胃潰瘍甚至胃癌的產生,但是現今治療胃幽門螺旋桿菌的藥物藥性已經漸漸下降產生抗藥性並且尚無新藥的開發,所以研發新的藥物來對抗胃幽門螺旋桿菌是迫在眉睫的事。我們所用來當作藥物設計的目標蛋白是在胃幽門螺旋桿菌中參與輔酶A生成反映途徑中的速率限制步驟的酵素全名叫磷酸泛酸醯基乙胺腺苷轉移酶,已有研究指出如將此蛋白的基因去除的話則會使得輔酶A的生成大量減少並影響微生物的生長。

我們過去已解出磷酸泛酸醯基乙胺腺苷轉移酶與輔酶A的複合體結構,為了想要了解磷酸泛酸醯基乙胺腺苷轉移酶與其受質是如何結合和那些胺基酸是有參與催化反應,我們將各個可能參與反應的氨基酸突變成丙胺酸,在進一步的利用酵素的逆反應動力學來去比較突變後的蛋白與原始蛋白活性的差異,以及利用圓二色光譜看二級結構是否有改變,最後利用X光繞射儀看突變後的蛋白其胺基酸與受質間的作用力是否有改變。目前在此研究中已初步篩選出對於磷酸泛酸醯基乙胺腺苷轉移酶與受質結合和催化非常重要的氨基酸。

H. pylori is a gram-negative and microaerophilic bacterium that infects more than 50% of the world population and increases the risk of developing gastric ulcer and stomach cancer. Antibiotics, e.g., amoxicillin and clarithromycin, and a proton-pump inhibitor have been used to treat H. pylori infection, but as H. pylori has become increasingly resistant to antibiotics, treatment often fails. It is important to find a new treatment or antibacterial drug targets to against H. pylori infection. Coenzyme A (CoA) is an essential cofactor and a major intracellular carrier of activated acyl groups in all living organisms. Phosphopantetheine adenylyltransferase (PPAT) from H. pylori is a penultimate, rate-limiting enzyme in the CoA biosynthesis pathway. The enzyme transfer adenylyl group from ATP to phosphorpantetheine, yielding dephospho-CoA and pyrophosphate. Recent study indicated that knock out the PPAT gene in Escherichia coli reducing its CoA levels and preventing bacterial growth. In addition, amino acid sequence and secondary structure between H. pylori and human PPATs are dissimilar. Therefore, PPAT is a potential antibacterial target. We have determined the complex structure of H. pylori PPAT with a CoA molecule (PDB ID: 3OTW). In H. pylori PPAT, the side chains of Thr10, Lys42, Arg88, and Tyr98 form hydrogen bonds with the CoA phosphate group. In contrast, fewer hydrogen bonds between other PPATs and CoA are observed. Interactions between Thr15, Gly17, Ile127, and Arg133 in H. pylori PPAT and CoA alter the orientation of the CoA adenine ring. To investigate the structural and kinetics mechanisms of substrate binding to the phosphopantetheine adenylyltransferase and its mutants from H. pylori, we used alanine-scanning mutagenesis, x-ray crystallography, biophysical, and steady-state kinetics of the reverse reaction approaches to answering these questions.

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