克雷伯氏肺炎桿菌中與致病機制相關的醣酵素之結構與反應機轉研究

克雷伯氏肺炎桿菌(Klebsiella pneumoniae) 為一革蘭氏陰性細菌。這種伺機性的病原體，在台灣所引發的典型症狀有原發性肝膿瘍 (primary liver abscess)、轉移性腦膜炎 (metastatic meningitis)、敗血症 (sepsis) 和眼內炎 (endophthalmitis)等。本論文研究的主要針對與克雷伯氏肺炎桿菌致病性相當重要的兩個目標蛋白進行研究，以瞭解其反應機制。第一個目標蛋白為6-磷酸葡萄糖酸脫氫酶 (6-phosphogluconate dehydrogenase, 6PGDH)，在Streptococcus pneumoniae 及 Streptococcus suis具有黏附因子(adhesion) 的功能，也在五碳糖磷酸途徑中 (pentose phosphate pathway) 參與了第三步驟的催化反應，其催化6-磷酸葡萄糖酸(6-phosphogluconate, 6PG) 之脫羧基反應，釋出二氧化碳以形成5-磷酸核酮糖 (ribulose 5-phosphate)，此反應需要一分子的輔酶NADP+參與反應，反應過程中會伴隨著NADPH的產生，做為還原反應的產物。第二個目標蛋白為尿嘧啶雙磷酸葡萄糖去氫酶 (UDP-Glucose dehydrogenase, Ugd)，其催化尿嘧啶雙磷酸葡萄糖 (UDP-glucose, UPG) 氧化反應生成尿嘧啶雙磷酸葡萄糖醛酸(UDP-glucuronic acid, UGA)，並伴隨著產生兩分子還原態輔酶NADH。尿嘧啶雙磷酸葡萄糖醛酸為4-amino-4-deoxy-L-arabinopyranose (L-Ara4N)生合成所必需的前驅物。藉由L-Ara4N在脂質多醣體 (LPS) 的lipid A上的修飾，使得細胞表面負電性降低，造成帶正電的多黏菌素 (polymyxin) 與克雷伯氏肺炎桿菌的細胞膜結合減少，進而讓克雷伯氏肺炎桿菌得以產生抗性，並保護革蘭氏陰性菌得以逃避抗菌胜肽 (cationic antimicrobial peptides) 的殺菌作用。研究結果發現第一個具有與大腸桿菌高同源性之致病菌克雷伯氏桿菌為來源之apo-form的結晶結構。以及結合受質、受質與輔酶 (NADPH) 及葡萄糖的三種大腸桿菌6-磷酸葡萄糖酸脫氫酶之高解析度複合結構。相較於未結合輔酶的單體，我們發現當結合輔酶NADPH至雙隅體其中之一的單體會誘發10度的旋轉及7埃的位移。因此，使得結合輔酶NADPH的單體比起未結合輔酶的單體，呈現出相對緊閉的構形。相較於他物種，我們進一步提出了結合輔酶時所產生相對的移動，進而影響輔酶結合區的構形的解釋。對6-磷酸葡萄糖酸脫氫酶，我們提出了一個反應物依序與相對應的胺基酸結合後，進而影響單體構形的機制；此外其對糖共軛體結合的差異性更提供了設計酵素抑制物相當重要的依據。對尿嘧啶雙磷酸葡萄糖去氫酶，本研究解出克雷伯氏肺炎桿菌尿嘧啶雙磷酸葡萄糖去氫酶單體的apo-form及末端帶有histidine-tag之高解析度結構。以及結合受質、受質與輔酶 (NADH) 及產物的三種克雷伯氏肺炎桿菌尿嘧啶雙磷酸葡萄糖去氫酶複合結構。在結合兩個產物分子的尿嘧啶雙磷酸葡萄糖去氫酶複合結構中，首次發現了一個不同於產物生成反應區的第二結合位。此結合位早已存在於結構表面，主要由帶正電的胺基酸組。相較於其他物種的尿嘧啶雙磷酸葡萄糖去氫酶結構，可得知K256 和 D257與第二個產物分子結合時會影響其存在的位置，並引發牽引的作用，造成參與催化反應的cysteine遠離了受質，形成了一個無法進行反應的構形。活性分析結果更進一步顯示了異位效應及產物競爭性抑制的現象，我們因而提出了一個負反饋機制來解釋結構的變化。

關鍵字

克雷伯氏肺炎桿菌； 6-磷酸葡萄糖酸脫氫酶尿嘧啶雙磷酸葡萄糖去氫酶藥物標的；抗生素失效

並列摘要

The Gram-negative bacterium, Klebsiella pneumoniae (Kp), is an opportunistic pathogen that mainly causes primary pyogenic liver abscess, metastatic meningitis, sepsis and endophthalmitis in Taiwan. The main thrust of this thesis work is the understanding of the reaction mechanisms of two target proteins associated with the pathogenesis of K. pneumoniae. One is 6-phosphogluconate dehydrogenase (6PGDH) that acts as a new cell wall adhesin in Streptococcus pneumoniae and Streptococcus suis, as well as the third enzyme of the pentose phosphate pathway, catalyzing the oxidative decarboxylation of 6-phosphogluconate to form ribulose 5-phosphate, along with the reduction of NADP+ to NADPH. The other is UDP-glucose dehydrogenase (Ugd), catalyzing the NAD+-dependent 2-fold oxidation of UDP-glucose (UPG) to produce UDP-glucuronic acid (UGA), a requisite precursor for the biosynthesis of 4-amino-4-deoxy-L-arabinopyranose (L-Ara4N), that allows K. pneumoniae to resist the antibiotic polymyxin and protect gram-negative bacteria from the bactericidal action of cationic antimicrobial peptides (CAMPs) by the cationic modification of phosphates of lipid A with L-Ara4N. Here we report the first apo-form crystal structure of the pathogenic K. pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH, and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10° rotation and resulting in a 7 Å movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme. For 6PGDH, we propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits and the novel glycoconjugate-binding ability of 6PGDH provide important implications for the design of selective inhibitors. For K. pneumoniae Ugd (KpUgd), we have determined crystal structures of KpUgd in the apo and C-terminal 6×histidine-tagged states at high resolution as well as complexes with substrate, substrate/NADH, and two product molecules. The binding of two UGA molecules to the KpUgd structure reveals for the first time that the second UGA occupied the pre-existing position of the positively charged surface pocket distinct from the active site. Superimpositions and comparisons of our KpUgd structures indicated that the second UGA interacted with K256 and D257, which in turn gives rise to the concomitant movement of the substrate-interacting cysteine and forms an inactive configuration. The kinetic studies showed that KpUgd exhibited allosteric effects and the competitive inhibition of product implicated that the catalysis follows a negative feedback mechanism.

並列關鍵字

Klebsiella pneumoniae ； 6-phosphogluconate dehydrogenase ； UDP-glucose dehydrogenase ； drug target ； antibacterial resistance

參考文獻

Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T., Warren, G.L., 1998. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905-921.

Adams, M.J., Ellis, G.H., Gover, S., Naylor, C.E., Phillips, C., 1994. Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism. Structure 2, 651-668.

Alvarez, D., Merino, S., Tomas, J.M., Benedi, V.J., Alberti, S., 2000. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates. Infect Immun 68, 953-955.

Baker, N.A., Sept, D., Joseph, S., Holst, M.J., McCammon, J.A., 2001. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 98, 10037-10041.

Bar-Peled, M., Griffith, C.L., Ory, J.J., Doering, T.L., 2004. Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans. Biochem J 381, 131-136.

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克雷伯氏肺炎桿菌中與致病機制相關的醣酵素之結構與反應機轉研究

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