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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 尿苷5？-二磷酸-N-乙酰葡萄糖胺（UDP-GlcNAc）是合成从治疗药物和疫苗到食品工业的各种聚糖和糖缀合物的必需供体。在自然界中，各种生物体采用不同且大多数复杂的途径来合成用于生物合成细胞壁、糖胺聚糖如透明质酸等的UDP-GlcNAc。UDP-GlcNAc的化学合成也复杂且昂贵。我们的最终目标是设计一种简单，经济和有效的酶促合成UDP-GlcNAc。为了实现这一点，我们使用以GlcNAc和ATP作为起始材料的双酶系统。GlcNAc在利用ATP的同时被称为NahK的酶转化为GlcNAc-1-P。GlcNAc-1-P在利用UTP的同时通过GlmU进一步转化为UDP-GlcNAc。 虽然GlmU已被结构和生物化学表征，NahK不是。本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。希望这些研究将最终导致开发一种更稳定和有效的酶，具有最佳的底物特异性。 NahK（B.longum）是中等分子量（~ 40 kD）的可溶性酸性酶。该蛋白属于蛋白激酶超家族，具有N-乙酰己糖胺激酶的功能。它是一种相对新颖和独特的酶，是第一个报道的用作葡萄糖型激酶的酶（参考文献#1），即，可以磷酸化葡萄糖及其类似物。 进行结晶尝试以获得该酶的结构。最终可以获得最大尺寸为40-50微米的小晶体。由于太小，它们只能在家用X射线源（5埃分辨率）上表现不佳。利用蛋氨酸营养缺陷型宿主菌获得含硒蛋氨酸蛋白。从这种硒蛋白中获得的晶体将用于MAD研究。