Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Abstract

UDP-glucose dehydrogenase (UGDH) is (6, 7) show that the reaction of UGDH involves two successive oxidations to convert the 6Ј-hydroxyl of UDP-glucose to a carboxylate, together with the reduction of 2 molecules of NAD ϩ to NADH. In animals, it constitutes the unique pathway for glucuronate formation (8). Because glucuronate is a component of glycosaminoglycans (GAGs), the mutation inactivation of UGDH (sugarless) abolishes GAG assembly and, consequently, abolishes GAG-dependent growth factor signaling (9 -11). The primary structure of the mammalian enzyme was obtained from the protein sequence of bovine UGDH (12). The human gene was also recently cloned and assigned to chromosome 4p15.1 (13). It contains 12 exons, extends over 26 kb, and has one major transcription start site (14).GAG chains of proteoglycans and hyaluronan are ubiquitous components of extracellular matrix and pericellular spaces. There is a growing body of information on the implication of GAGs in cell behavior, including signal transduction, cell proliferation, spreading, migration, and cancer growth and metastasis (15-17). GAG synthesis is influenced by cytokines and growth factors. Transforming growth factor-␤ is the most potent stimulator of proteoglycan and GAG synthesis, including that of hyaluronan. Its action, however, depends on the cell type (18). The synthesis of GAGs is also modulated by oxygen cell status. Hypoxic endothelial cells and lung fibroblasts enhanced the heparan sulfate/chondroitin sulfate ratio, which led to an increase of basic fibroblast growth factor reactivity on the cell surface (19,20). It was also shown that the level of intracellular UDP-glucuronate could influence GAG synthesis (8). Interestingly, the human UGDH was shown to be an early response gene after interleukin-1 treatment of ocular fibroblasts (21), as well as an early androgen response gene in breast cancer (22).Although a clone of human UGDH has been reported (13, 15), specific catalytic residues are not yet available. Therefore, further characterization of the active sites of human UGDH is needed to elucidate the physiological nature of the UGDH. In the present study, we have identified an NAD ϩ -binding site using photoaffinity labeling and cassette mutagenesis to gain a deeper insight into the structural basis of human UGDH. For this study, a 1509-base pair gene that encodes human UGDH has been chemically synthesized and expressed in Escherichia coli as a soluble protein. Identification of the nucleotide-binding sites of a variety of proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert * This work was supported by Grant 03-PJ1-PG3-20900-0047 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea (to S.-W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)

Conformational change upon product binding to Klebsiella pneumoniae UDP-glucose dehydrogenase: A possible inhibition mechanism for the key enzyme in polymyxin resistance

“…S1) (Breazeale et al, 2005;Huh et al, 2004). UGA also serves as a precursor for synthesis of bacterial antiphagocitic CPS, colanic acid (M-antigen), plant cell walls, and mammalian glycosaminoglycans (Hung et al, 2007;Lacour et al, 2008), and it is critical to bacterial virulence (Jiang et al, 2010) as required for the biosynthesis of extracellular polysaccharide.…”

“…It is emerging as a major causative agent of multi-drug resistant infections and pyogenic liver abscess frequently complicated by metastatic meningitis or endophthalmitis (Fang et al, 2004;Wu et al, 2009). Expression of two essential virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), in K. pneumoniae clinical isolates (Huh et al, 2004) allows it to escape from host innate immune responses (Easley et al, 2007). The LPS modifications with 4-amino-4-deoxy-L-arabinopyranose (L-Ara4N) further confer K. pneumoniae resistance to the antibiotic polymyxin (Helander et al, 1996), a member of cationic antimicrobial peptides (CAMPs).…”

Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase

“…Recombinant wild-type hUGDH was purified and the activity verified spectrophotometrically by measuring the reduction of NAD + in the presence of UDP-glucose [39]. A deletion mutant was generated that lacked amino acids 488–494, which was also purified via this protocol [32].…”

Induced and repressed genes after irradiation sensitizing by pentoxyphylline

“…59 The enzyme UDPGDH converts UDPglucose to UDP-glucuronate, which is critical in the carbohydrate metabolism. 60 The clone 1.32, in the Ref_Seq database, shows the best homology to the FTL gene coding the ferritin, light chain polypeptide. Ferritin is composed of 24 chains, is spherical and contains a central cavity into which the polymeric ferric iron core is deposited and is the major intracellular iron storage.…”