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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尿苷5？-二磷酸-N-乙酰氨基葡萄糖(UDP-GlcNAc)是合成多种糖链的重要供体，广泛应用于从治疗药物、疫苗到食品工业等领域。在自然界中，各种生物采用不同且最复杂的途径来合成用于细胞壁生物合成的UDP-GlcNAc，以及透明质酸等糖胺聚糖等。UDP-GlcNAc的化学合成也是复杂和昂贵的。我们项目的最终目标是设计一种简单、经济和高效的UDP-GlcNAc的酶合成方法。为了实现这一点，我们使用了以GlcNAc和ATP为起始原料的双酶系统。在利用ATP的同时，一种名为NAHK的酶将GlcNAc转化为GlcNAc-1-P。GlcNAc-1-P在利用UTP的同时，被GlmU进一步转化为UDP-GlcNAc。虽然GlmU已经在结构和生化上得到了表征，但NAHK还没有。当前项目的重点是研究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出一种更稳定、更高效、具有最佳底物专一性的酶。 NAHK(B.long um)是一种中等相对分子质量(~40kD)的可溶性酸性酶。该蛋白属于蛋白激酶超家族，具有N-乙酰氨基己糖的功能。它是一种相对新颖和独特的酶，因为它是第一个被报道的充当葡萄糖型激酶的酶(参考文献1)。#1)，即可以使葡萄糖及其类似物磷酸化。为了获得该酶的结构，进行了结晶尝试。最终可以得到最大尺寸为40-50微米的小晶体。由于它们太小，在家用X射线源(分辨率为5埃)下的衍射率很低。利用蛋氨酸营养缺陷型宿主菌株获得了含硒蛋氨酸的蛋白质。从这种硒蛋白中获得的晶体将被尝试用于MAD研究。

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.