尿嘧啶雙磷酸葡萄糖去氫酶 (UDP-glucose dehydrogenase, UGDH)催化尿嘧啶二磷酸葡萄糖 (UDP-glucose)氧化反應變成尿嘧啶雙磷酸葡萄糖醛酸(UDP-glucuronic acid)，伴隨著產生兩分子的NADH。尿嘧啶雙磷酸葡萄糖醛酸扮演很多重要的生理角色，雖然其生化特性從人類到細菌已經被廣泛的研究，但是它的生化特性對於綠膿桿菌的生物意義目前仍不清楚。在這篇論文當中，我選殖、表現、以及純化兩個基因PA2022和PA3559的產物，證實此兩個基因的功能皆為尿嘧啶雙磷酸葡萄糖去氫酶。對於尿嘧啶雙磷酸葡萄糖而言，PA2022和PA355的Km分別是0.061 mM 和0.646 mM；對於另一個受質NAD+，PA2022和PA355的Km分別是0.379 mM and 1.003 mM。除此之外，PA3559對於尿嘧啶雙磷酸葡萄糖的專一性比PA2022高。PA2022除了會利用尿嘧啶雙磷酸葡萄糖之外，還會利用TDP-glucose和 UDP-N-acetylglucosamine。凝膠管柱層析實驗結果顯示PA2022和PA3559在水溶液中為雙體結構。我也構築綠膿桿菌尿嘧啶雙磷酸葡萄糖去氫酶的缺損株。其中PA2022和PA3559單一基因缺損株分別稱RJ 101與RJ102，而雙基因缺損株則稱RJ103。藉由比較野生株與突變株，可發現尿嘧啶雙磷酸葡萄糖去氫酶可能參與抗藥性和生物膜形成。RJ103缺損株對於抗生素chloramphenicol、cefotaxime和 ampicillin的敏感度增加;除此之外，它的爬行能力降低，形成生物膜的量和厚度都比野生株低。本研究顯示尿嘧啶雙磷酸葡萄糖去氫酶在綠膿桿菌中可藉由合成玻尿酸前趨物:尿嘧啶雙磷酸葡萄糖醛酸來調節胞外多醣體的合成與致病機轉。

UDP-glucose dehydrogenase (UGDH) catalyzes the NAD+-dependent twofold oxidation of UDP-glucose to yield UDP-glucuronic acid, a substrate for polysaccharide biosynthesis in many strains of pathogenic bacteria. Although the functions and kinetic properties of UGDH have been studied extensively in organisms ranging from bovine to bacteria; the biochemical properties of UGDH in Pseudomonas aeruginosa PAO1, an important pathogen, is still unknown. In this study, we have cloned, expressed, and affinity-purified the product of two open reading frame, PA2022 and PA3559, which have been annotated as nucleotide sugar dehydrogenase and determined their kinetic parameters, substrate specificities, and oligomeric states. The Km of PA2022 and PA3559 for UDP-glucose are 0.061 mM and 0.646 mM, while the Km of these two enzymes for NAD+ is 0.379 mM and 1.003 mM, respectively. Besides, the substrate specificity of PA2022 is quite different from that of PA3559 that is more specific to UDP-glucose. PA2022 can also utilize TDP-glucose and UDP-N-acetylglucosamine with the one-third velocity of UDP-glucose. The gel filtration data showed that both the UGDH are active as dimer in solution. Comparison between the wild type PAO1 and mutants revealed that the possible functions of the UGDHs in P. aeruginosa included the participation in antibiotic resistance and biofilm formation. The PA2022-PA3559 double mutant RJ103 was more susceptible than wild type PAO1 to chloramphenicol, cefotaxime, and ampicillin. Moreover, its twitching motility was impaired and it produced substantially thinner biofilm than wild type PAO1. The data suggests that the possible biological role of UGDH, which syntheses UDP-glucuronic acid as a precursor for hyaluronic acid, involves in the biosynthesis of critical exopolysaccharide components and may regulate P. aeruginosa pathogenesis.

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