X-ray Studies of Sugar-Modifying Enzymes

描述（由申请人提供）：碳水化合物的生物学重要性怎么强调都不为过，因为它们几乎是每一个生理过程的必需元素，代表了生命系统中最丰富的生物分子。除了提供代谢能量的作用外，碳水化合物还参与广泛的生物过程，包括免疫反应、细胞间相互作用、受精和细胞粘附等。在过去的11年里，这笔资金支持了对构成半乳糖代谢Leloir途径的四种酶的x射线结构和生化分析。编码这些酶的基因缺陷可导致疾病状态，统称为半乳糖血症。这些疾病的临床严重程度差别很大，取决于受影响的基因，以及由此导致的酶损伤程度。通过这个实验室的努力，所有这些酶的结构现在都是已知的，其中两个在当前的资助期内得到了解决。此外，近年来，该基金还支持了一些酶的结构分析，这些酶参与了不寻常的脱氧糖的合成：tyvelose、colitose和去糖胺。Tyvelose和colitose是3,6-二脱氧己糖，存在于一些革兰氏阴性细菌的o抗原中，并被推测在这些细菌的致病性中起作用。去糖胺是一种3-（二甲胺）-3,4,6-三脱氧己糖，存在于某些大环内酯类抗生素中，如常用的红霉素。在聚酮类抗生素中加入不寻常的脱氧糖，如d -去糖胺，可提供或增强其生物活性。这种竞争性更新的目标本质上是双重的：(i)继续我们对脱氧糖生物合成中涉及的酶的结构和功能的研究，（ii）启动对酿酒酵母中调节GAL遗传开关编码基因转录的蛋白质的结构研究。这项新研究是我们过去对Leloir通路研究的自然延伸。具体来说，GAL基因开关编码Leloir通路酶的基因。到目前为止，已经确定了控制这些基因转录的三个关键成分：GalSp， GalSOp和Gal4p，这些将成为结构/功能研究的目标。对于所有计划的研究，将采用x射线晶体学、定点诱变和生化技术的结合。综上所述，这些研究将为糖代谢和真核生物转录调控提供新的信息。

DESCRIPTION (provided by applicant): The biological importance of carbohydrates cannot be overstated for they are essential elements in nearly every physiological process and represent the most abundant bio-molecules in living systems. Apart from their role in providing metabolic energy, carbohydrates are involved in a wide range of biological processes including the immune response, cell-cell interactions, fertilization, and cell adhesion, among others. This grant has supported over the last 11 years both x-ray structural and biochemical analyses of the four enzymes that constitute the Leloir pathway for galactose metabolism. Defects in the genes encoding these enzymes can lead to diseased states referred to collectively as galactosemia. Clinical severity of these disorders varies widely, depending upon the gene affected, and the resulting degree of enzyme impairment. Through the efforts of this laboratory, the structures of all of these enzymes are now known, two of which were solved during the current funding period. Additionally, in recent years, this grant has supported the structural analyses of enzymes involved in the synthesis of the unusual deoxysugars: tyvelose, colitose, and desosamine. Tyvelose and colitose are 3,6-dideoxyhexoses that occur in the O-antigens of some Gram- negative bacteria and have been speculated to play a role in the pathogenicity of these organisms. Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. The addition of unusual deoxysugars such as D-desosamine to such polyketide antibiotics provides or enhances their biological activity. The goals of this competitive renewal are twofold in nature: (i) to continue our research on the structure and function of enzymes involved in deoxysugar biosynthesis, and (ii) to initiate structural studies on the proteins in Saccharomyces cerevisiae that regulate transcription of the genes encoded by the GAL genetic switch. This new study is a natural extension to our past research on the Leloir pathway. Specifically, the GAL genetic switch encodes the genes for the enzymes of the Leloir pathway. Thus far three key components have been identified in controlling transcription of these genes: GalSp, GalSOp, and Gal4p and these will be targeted for structural/functional studies. For all of the planned investigations, a combination of x-ray crystallographic, site-directed mutagenesis, and biochemical techniques will be employed. Taken together, these studies will provide new information with respect to both sugar metabolism and eukaryotic transcriptional regulation.