Study of enzymes involved in the syntheses of dinucleotide sugar building blocks GDP-fucose, UDP-glucose, and UDP-glucuronate for capsular polysaccharide from Klebsiella pneumoniae (NTUH-K2044)

• Main Authors: Chun-yen Lee, 李俊彥
• Other Authors: Tsung-Lin Li
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/69714476330489854199

碩士 === 國立臺灣海洋大學 === 食品科學系 === 95 === Klebsiella pneumoniae is a Gram-negative bacterium, which is the main cause of hospital-acquired infections. Klebsiella pneumoniae can be funther classified into invasive and noninvasive on the basis of their mucoviscosity. The diseases caused by the invasive K. pneumoniae in Taiwan exist some common symptoms, such as primary liver abscess, sepsis, meningitis and endophthalmitis. It has been proposed that capsular polysaccharide (CPS) and the resulting capsular serotype demonstrate certain relationships with the pathogenicity. In this thesis, genes that are involved in the biosynthesis of capsular polysaccharide from K. pneumoniae NTUH-K2044 were studied. We hope with the efforts input we can better understand the biological roles of these genes, so that we would be able to provide knowledge-based preventions and treatments to the K. pneumoniae infections. There are 28 open reading frames involved in the gene cluster of the CPS biosynthesis. The gene cluster can be further divided into three groups by their functionality: (1) genes for dinucleotide phosphate sugar biosynthesis, including GDP-fucose and UDP-glucuronate, (2) genes for glycosyltransferation, and (3) genes for assembly regulation, and export for the CPS. This study was mainly focused on the biosyntheses of GDP-fucose and UDP-glucuronate. Two pathways were proposed, which contain 9 gene products. They are KP3699, KP3701, KP3702, KP3703, KP3704, KP3708, KP3709, KP3711 and KP3726. The recombinant hetrologous protein technology was applied for gene cloning, protein expression, and purification. A series of experiments were conducted in vitro to identify and characterize their respective biological roles. KP3726, KP3701, and KP3699 were experimentally demonstrated to be related to the biosyntheses of UDP-glucose, UDP-glucuronic acid, and UDP-galacturonic acid. The molecular weight of KP3726 is about 32kDa. It can catalyze the formation of UDP-glucose in the presence of UTP, glucose-1-phosphate, and Mg2+, so KP3726 was confirmed to be a glucose-1-phosphate uridylyltransferase. The molecular weight of KP3701 is about 43kDa. The enzyme can convert the production of UDP-glucuronate from UDP-glucose. NAD+ is needed for the reaction with a concomitant production of NADH. Therefore, KP3701 was confirmed as a UDP-glucose dehydrogenase. Once UDP-glucuronate was produced, it can be further catalyzed by KP3699 (37kDa) into UDP-galacturonate. Although KP3699 was confirmed as a UDP-glucuronic acid 4- epimerase, its role in the CPS biosynthesis is still unknown. KP3702, KP3703, and KP3711 were demonstrated to be related to the biosyntheses of GDP-mannose and the precursor of GDP-fucose. KP3702 (50kDa) is able to converse mannose-6-phosphate to mannose-1-phosphate, wherein Mg2+ is needed. So, KP3702 was confirmed as a phosphomannomutase. KP3703 (52kDa) catalyzes mannose-1-phosphate into GDP-mannose with the consumption of a molecule of GTP. Therefore, KP3703 was confirmed as a GDP-mannose pyrophosphorylase. KP3711 (43kDa) can catalyze GDP-mannoses to become GDP-4-keto-6-deoxy-mannose, and Mg2+ is also needed in the reaction. KP3711 was confirmed as a GDP-mannose 4, 6- dehydratase. This is the first step of GDP-fucose biosynthesis from GDP-mannose. KP3709 is thought to be a bifunctional enzyme. It presumably catalyzes the production of GDP-fucose through sequential isomerization and reduction reactions. In addition, KP3704 and KP3708 may participate in the regulation of GDP-mannose and GDP-fucose. KP3704 (51kDa) has be confirmed as a gluconate-6-phosphate dehydrogenase can oxidize gluconate-6-phosphates into ribulose-5- phosphate with the production of NADPH. NADPH is required in the reaction for GDP-fucose production. KP3708 (19kDa) Can hydrolyze GDP-mannose into GDP and mannose, wherein Mg2+ is needed in the reaction. It balances the productions of GDP-mannose and GDP-fucose. KP3708 was therefore confirmed as a GDP-mannose mannosyl hydrolase.