鮑氏不動桿菌 oxacillinase-66 Ile-129 以及 Trp-222 在酵素活性與受質專一性的探討

Study of the enzyme activity and substrate specificity of oxacillinase-66 Ile-129 and Trp-222 in Acinetobacter baumannii

方獻郎(Xian-Lang Fang)

指導教授 ： 陳凌雲

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2011年)

摘要

Acinetobacter baumannii (A. baumannii) 鮑氏不動桿菌，屬於革蘭氏陰性的球桿菌，為伺機性致病菌。目前臨床以carbapenems類抗生素中的imipenemu以及meropenem來對抗multidrug resistant A. baumanni (MDRAB)，但是A. baumannii可產生class D β-lactamases的oxacillinases (OXAs) 分解抗生素而產生抗藥性。目前A. baumannii的OXAs主要有OXA-23 like、OXA- 40/24 like、OXA-51 like以及OXA-58 like 4個亞群，其中OXA-51 like為最大的亞群，但酵素活性較弱。Yang等人在2010發表的論文中發現有7株單獨僅帶有OXA-51 like基因的MDRAB菌株，但並未進行DNA序列分析以瞭解是否是基因的突變而增強酵素活性。本實驗室取得4株上述單獨帶有OXA-51 like基因的MDRAB菌株 (ABMT1-4)，以DNA選殖得知ABMT-1與ABMT-4皆為過去已發表的OXA-172 (I129V, W222L)。另外，也從臨床取得23株carbapenem resistance A. baumannii (ABLR1-23)，4株carbapenem susceptible A. baumannii (ABLS1-4)，總共27株，以多重聚合酶連鎖反應發現抗藥性菌株只有ABLR-13單獨帶有OXA-51 like基因，以DNA選殖得知ABLR-13為一個新的突變，命名為OXA-66LR13 (I129V)。接著，以胺基酸多重序列比對所有已知的OXA-51 like基因，得知OXA-83在Ile-129突變成leucine，以定位突變法我們取得OXA-83 (I129L) 以及OXA-83L (I129L, W222L) 兩個突變蛋白。將上述4種突變 (OXA-66LR13, OXA-83, OXA-83L, OXA-172) 以及OXA-66重組蛋白純化出後，進行酵素水解受質的imipenem以及meropenem以及ertapenem的相對比活性分析，發現I129L對酵素活性的提升遠比I129V高，W222L的突變對酵素活性則有加乘的效果。以酵素動力學發現W222L會增加OXA-83蛋白對受質的親和力。最後，以OXA-24 X-ray晶體結構為模版，使用Swiss-PdbViewer電腦軟體模擬OXA-51 like蛋白催化中心入口結構，發現I129V以及I129L都可使得入口變大，而leucine碳原子位於側鏈第2個碳，增加催化中心底部平台的空間來容納並支撐受質。W222L的突變會使得催化中心入口上緣的疏水性障壁開口更為緊密，使受質更能穩固的留在催化中心接受酵素水解。總結，在現今細菌抗藥性不斷地增加下，本研究的實驗結果對於未來研究細菌抗藥性的成因以及研究新藥對抗抗藥性強的細菌，提供一個非常好的研究模式。

關鍵字

鮑氏不動桿菌

並列摘要

Acinetobacter baumannii is gram-negative coccobacillus and opportunistic pathogens. Carbapenems (imipenem, meropenem) antibiotics are used to combat multidrug resistant A. baumannii (MDRAB). Resistance to carbapenems may due to the production of class D β-lactamases, oxacillinases (OXAs), which can hydrolyze β-lactam antibiotics. At present, A. baumannii OXAs mainly has four groups: OXA-23 like subgroup, OXA- 40/24 like subgroup, OXA-51 like subgroup and OXA-58 like subgroup. The OXA-51 like subgroup is the largest subgroup, but has the weakest enzyme activity. According to the paper published, Yang (Yang et al., 2010) they found 7 out of the 192 isolates (3.65%) had only one carbapenemase subgroup gene (OXA-51 like), but not for DNA sequence analysis to find out whether the gene mutation contribute to the increase of enzyme activity. Our laboratory has obtained 4 strains from the seven isolates (ABMT1-4). By DNA cloning, we knew that ABMT-1 and ABMT-4 were OXA-172 (I129V, W222L) has been published. We also obtained 23 carbapenem resistance A. baumannii clinical isolates (ABLR1-23) and 4 carbapenem susceptible A. baumannii (ABLS1-4) clinical isolates, total 27 isolates. We found only ABLR-13 had only one carbapenemase subgroup gene (OXA51-like) by multiplex PCR. For DNA cloning, we find that ABLR-13 was a novel mutation named OXA-66LR13 (I129V). The multiple sequence alignment of all known OXA-51 like proteins, we found that isoleucine at position 129 of OXA-83 was replaced by leucine. According to this finding, we construct two site-directed mutants, one with single mutation I120L (named OXA-83) and the other with double mutation I120L, W222L (named OXA-83L). We purified the recombinant protein of four mutants (OXA-66LR13, OXA-83, OXA-83L, OXA-172) and OXA-66, and analyzed their relative specific activity to the substrates, imipenem, meropenem and ertapenem. We found that I129L could improve the enzyme activity than I129V, and W222L mutation for the enzyme activity has additive effects. We also found W222L could increase the affinity of OXA-83 protein for the substrate meropenem by the enzyme kinetics. Finally, we took the OXA-24 X-ray crystal structure as a template and progressed structure prediction of OXA-51 like protein active site entrance by simulation software: Swiss pdbviewre. We found that I129V and I129L could make the entrance larger, and I129L also could improves the substrate held in the active site, because of the carbon atom is located on the 2nd carbon of the leucine side chain. W222L mutation made the open space of the hydrophobic barrier which above the active site entrance closer, to bring the substrate to hold more stable for enzyme hydrolysis in the active site. Summary, the antibiotic resistance of bacteria is increasing today; the results of this study provide a good research model for the causes of the bacterial antibiotics resistance and finding new drugs against resistant bacteria in the future.

並列關鍵字

Acinetobacter baumannii ； carbapenem ； oxacillinase

參考文獻

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