INVESTIGATING STRUCTURE AND SUBSTRATE-BINDING OF A NOVEL GLUCO-TYPE KINASE NAHK

研究新型葡萄糖型激酶 NAHK 的结构和底物结合

• 批准号: 8171989
• 负责人: Peng Wang
• 金额: $ 0.36万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171989
• 关键词: Acetylglucosamine; Anabolism; Binding; Cell Wall; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Development; Dimensions; Economics; Engineering; Enzymes; Food Industry; Funding; Glucose; Glycoconjugates; Glycosaminoglycans; Goals; Grant; Home environment; Hyaluronic Acid; Institution; Lead; Medicine; Methionine; Molecular Weight; N acetylhexosamine; N-acetylglucopyranosylamine; Nature; Organism; Pathway interactions; Phosphotransferases; Polysaccharides; Protein Kinase; Proteins; Reporting; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Selenomethionine; Source; Structure; Substrate Specificity; System; Therapeutic; United States National Institutes of Health; Uridine; Vaccines; analog; chemical synthesis; enzyme structure; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Uridine 5?-diphospho -N-acetylglucosamine (UDP-GlcNAc) is an essential donor for synthesis of a diverse array of glycans & glycoconjugates used in fields spanning from therapeutic medicine and vaccines to food industry. In nature, various organisms employ different and mostly complicated pathways to synthesize UDP-GlcNAc for biosynthesis of cell walls, glycosaminoglycans such as hyaluronic acid, etc. The chemical synthesis of UDP-GlcNAc is also complicated and costly. The final goal of our project is to engineer a simple, economic and efficient enzymatic synthesis of UDP-GlcNAc. In order to accomplish this we are using a two enzyme system with GlcNAc and ATP as starting material. GlcNAc is converted to GlcNAc-1-P by an enzyme called NahK while utilizing ATP. GlcNAc-1-P is further converted to UDP-GlcNAc by GlmU while utilizing UTP. Whereas GlmU has been characterized structurally and biochemically, NahK is not. The specific focus of current project is to investigate the structure of NahK and study its binding with various substrates and substrate analogs. It is hoped that these studies will eventually lead to development of a more stable and efficient enzyme with optimum substrate specificity. NahK (B.longum) is a soluble acidic enzyme of moderate molecular weight (~ 40 kD). The protein belongs to protein kinase superfamily & functions as N-acetylhexosamine kinase. It is a relatively novel and unique enzyme in being the first reported enzyme that acts as a gluco-type kinase (ref. #1) i.e., can phosphorylate glucose and its analogs. Crystallization attempts were undertaken to obtain structure of this enzyme. Small crystals of 40-50 microns in largest dimension could finally be obtained. Being too small they could diffract only poorly at the home X-ray source (5 angstrom resolution). Selenomethionine containing protein was obtained by using methionine auxotrophic host strain. The crystals obtained from this Se-protein would be tried for MAD studies.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 尿苷5？-Diphospho -N-乙酰葡萄糖胺（UDP-GLCNAC）是合成各种聚糖和聚糖阵列的必不可少的供体，用于从治疗药物和疫苗到食品行业的田野中使用的多种多样的供体。在本质上，各种生物使用不同且主要是复杂的途径来合成UDP-GLCNAC，用于细胞壁的生物合成，透明质酸等糖胺聚糖等。UDP-GLCNAC的化学合成也很复杂且昂贵。我们项目的最终目标是设计一种简单，经济和有效的酶促合成UDP-GLCNAC。为了实现这一目标，我们将两个酶系统与GlcNAC和ATP用作起始材料。使用ATP时，GlcNAC通过称为NAHK的酶转化为GlcNAC-1-P。使用UTP时，GlcNAC-1-P通过GLMU进一步转换为UDP-GLCNAC。 尽管GLMU在结构和生化上都被表征，但NAHK却没有。当前项目的具体重点是研究NAHK的结构，并研究其与各种底物和底物类似物的结合。希望这些研究最终能够以最佳的底物特异性来开发更稳定和有效的酶。 NAHK（B. longum）是一种中等分子量（〜40 kD）的可溶性酸性酶。该蛋白质属于蛋白激酶超家族，并用作N-乙酰己糖胺激酶。这是一种相对新颖且独特的酶，它是第一个充当葡萄糖型激酶（参考＃1）的酶，即可以磷酸化葡萄糖及其类似物。 进行了结晶尝试以获得该酶的结构。最终可以获得最大尺寸的40-50微米的小晶体。太小了，他们只能在Home X射线源（5 Angstrom分辨率）衍射。通过使用蛋氨酸营养营养宿主菌株获得含硒蛋白的蛋白质。从这种蛋白质蛋白获得的晶体将尝试进行疯狂研究。