王怡鈞Yi-Chun Wang林元鈞Yuan-Chun Lin生物醫學科學學系碩士班2026-03-189999-12-312014https://handle.ncl.edu.tw/11296/2m25x6https://scholars.csmu.edu.tw/handle/123456789/5876臨床上使用Carbapenem (β-lactam)類抗生素，例如Imipenem (ipm)、Meropenem (mpm)等，對付具有Oxacillinase (Class D β-lactamases)的感染性菌株。Acinetobacterbaumannii (AB菌)有OXA-23 like、OXA-24 like、OXA-51 like及OXA-58 like這四種亞群，而我們實驗中主要用到的OXA-66屬於OXA-51 like，是AB菌與生俱有的基因。從已解開的OXA-24晶體結構得知，受質會從通道狀的入口進入活化中心。OXA-66與OXA-24為同源蛋白，其相對應胺基酸位置129 Ile就位於通道狀的入口處，與我們合作的實驗室發現當OXA-66 I129點突變時，分解抗生素的活性會隨著改變。為了更確定OXA-66 I129位置胺基酸所扮演的角色，合作實驗室以基因選殖與點突變取得OXA-66及其位置I129突變為疏水性胺基酸Leu、Val、Ala的重組蛋白。然後觀察這些重組蛋白與受質ipm及mpm作用時活性上變化。結果發現對於受質ipm作用時Vmax相對值比較由大到小為381%(Leu)、155%(Ala)、115%(Val)、100%(Ile)，可以觀察到I129L上升倍數最高。而對於受質mpm作用時Vmax相對值比較由大到小為224%(Leu)、104%(Ala)、100%(Ile)、80%(Val)，可以觀察到I129L上升倍數最高，I129V最低。但合作實驗室從生化實驗上得到這些數據卻無法解釋為什麼129位置胺基酸的改變會造成活性上的變化，於是我們從結構上來協助他們解釋活性變化的原因。目前只有OXA-24的結構被解出來，而我們透過序列比對發現OXA-24與OXA-66的序列非常相似，於是我們使用PDB Viewer對OXA-24的結構進行突變來模擬OXA-66，為了確認模擬的結構是否正確，於是我們使用Modeller這個套裝軟體來模擬OXA-66的結構，經過比對發現這兩種方法所模擬出的結構是不相同的，所以我們將序列送到Swiss-Model(http://swissmodel.expasy.org/)請他們模擬，而他們模擬出的結構與我們使用Modeller模擬出的結構是一樣的，於是我們使用Modeller模擬出的OXA-66來進行接下來的實驗。我們使用Modeller模擬出OXA-66、OXA-66 I129L、OXA-66 I129A、OXA-66 I129V，觀察結構發現129位置胺基酸的改變會使129位置到Lys83之間的距離產生變化，因為129位置胺基酸是疏水性胺基酸，會降低Lys83的pKa值，使-NH3釋放質子接受CO2而形成羧化態，若129位置胺基酸與Lys83距離較近，水分子較不易進入Lys83周圍空間中，使Lys83較不易進行去羧化反應，而較利於進行水解反應，相反的若距離較遠水分子易進入Lys83周圍空間中，使Lys83去羧化，較不利進行水解反應[1]。根據另一篇文獻中提到[2]，Class D β-lactamases酵素與β-lactam抗生素作用時，活性中心Serine上的OH基會與β-lactam環上的碳結合，接著水分子進入而進行水解反應，因此我們推測活性中心Serine與β-lactam環結合的難易度會影響到Class Dβ-lactamases水解β-lactam的能力，於是我們使用AutoDock將OXA-66與其突變種來對ipm與mpm進行docking，觀察結果發現OXA-66 129位置胺基酸的改變會造成活性中心空間結構的改變，使得抗生素以不同的形態進入活性中心，因而影響活性中心Serine與β-lactam環結合的難易度，造成活性的變化。由以上結果推論OXA-66 129位置的胺基酸改變會對結構造成變化，這些變化會導致抗生素以不同的形態與活性中心結合，也會影響到Lys83的羧化反應，這些現象都會影響酵素催化受質的能力。Clinical application of Carbapenem( β-lactam) antibiotics including Imipenem(ipm) and Meropenem(mpm) to deal Oxacillinase(Class D β-lactamases).Acinetobacterbaumannii has four subgroups, which are OXA-23 like、OXA-24 like、OXA-51 like、and OXA-58 like. During the experiment, we mainly use OXA-66 that belongs to OXA-51 like and it is an inherent gene in Acinetobacterbaumannii. According to the crystal structure of OXA-24, substrate will enter the active site through a tunnel-like entrance. OXA-66 and OXA-24 are homologous protein, and its corresponding amino acid site-129 Ile is located at tunnel-like entrance. Collaboration lab found when I129 of OXA-66 mutates;the decomposition antibiotic''s activity will change as well. In order to explore the role of I129 of OXA-66, collaboration lab expressed and purified the OXA-66 and three substitution mutants at I129 with hydrophobic amino acids Leu, Val, and Ala, individually.All recombinant proteins were subjected to enzymatic assay with ipm and mpm.As a result, we found that when substrate imp is under function, Vmax''s descending contrast relative value is 381% (Leu), 155%(Ala), 15% (Val), 100% (Ile). It shows that I129L has the largest rise. On the other hand, when substrate mpm is under function, Vmax''s descending contrast relative value is 224% (Leu), 104% (Ala), 100% (Ile), 80% (Val). It shows that I129L has the largest rise and I129V has the lowest rise. However, even though collaboration lab obtains the data from the biochemical test, but they cannot explain why site-129 amino acid''s change will cause the alteration of the activity. Thus, we assist them by explaining the reason of the activity alteration through its structure.We used Modeller to simulate the structure of OXA-66, OXA-66I129L, OXA-66I129A, and OXA-66I129V. Then, we performed docking simulation with ipm and mpm to the OXA-66 and its mutants. Finally, we found that the change of amino acid of OXA-66 I129 will cause changes in the spatial structure of the active site. We found out when the ipm works, the S80 of OXA-66 I129L is closest with the carbon on the β-lactam of ipm and the activity is highest, and the S80 of OXA-66 I129 is farthest with the carbon on the β-lactam of ipm and the activity is the lowest. And when the mpm works, the S80 of OXA-66 I129L is closest with the carbon on the β-lactam of mpm and the activity is highest, and the S80 of OXA-66 I129V is farthest with the carbon on the β-lactam of mpm and the activity is lowest. This is consistent with the documents which pointed that when the enzyme Class D β-lactamases works with antibiotics β-lactam, the OH of serine will combine with the carbon on the β-lactam. Then, the water molecule get into and doing hydrolysis. Therefore, the difficult of combine with serine and β-lactam will influence the capacity of Class D β-lactamases to hydrolyze β-lactam.In 2012 Jennifer S.B. et. al. found that the 129 hydrophobic amino acids will reduce the pKa values of the K83, and it made -NH3 release proton to receive CO2. The degree of carboxylation of K83 will influence the capacity of Class D β-lactamases to hydrolyze β-lactam. From the result of docking, we can know that no matter ipm or mpm, the L129 and K83 are closest and the activity is highest. This is due to the hydrophobic nature of Leu and it is the closest to K83, it made K83 has smaller pKa value and much easier to carboxylate. Therefore, we infer that position 129 of OXA-66 may influence the degree of hydrophobicity of K83 then the capacity of carboxylation of K83.According to the result, we infer that the amino acid change of OXA-66 at position 129 will made change to the structure. These changes will lead antibiotics to combine with activity center by different form, and it also affects the degree of carboxylation of K83. All of these phenomenons influence the enzyme activity.thesis