STRUCTURAL STUDIES ON BETA-LACTAMASE ENZYMES

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。 β-内酰胺类抗生素是临床上最常见的抗生素，占全球抗菌药物总消费量的60%以上。它们包括青霉素类、头孢菌素类、单环内酰胺类、青霉烯类和碳青霉烯类，市场上有50多种这类抗生素。细菌对β-内酰胺抗生素的耐药性的主要机制是产生β-内酰胺酶，即在两步过程中水解β-内酰胺的保守四元环的酶。 首先，β-内酰胺环打开，加合物共价结合到活性位点丝氨酸残基上（酰化步骤）。然后，该加合物被高度保守的谷氨酸残基（Glu 166）固定的水分子脱酰，释放出药物的无活性青霉酸衍生物。 我们目前正在研究两种最近发现的β-内酰胺酶。 第一个，命名为GES-1（圭亚那扩展频谱，以其首次分离的国家命名），最初于2000年描述。 这种超广谱β-内酰胺酶（ESBL）与其他A类β-内酰胺酶的亲缘关系很远，对青霉素类和第一代、第二代和一些第三代头孢菌素（如头孢他啶）产生耐药性，但对单环内酰胺类和碳青霉烯类不产生耐药性。 自2000年以来，已经描述了来自不同地理位置的9种GES型酶（GES-1 - GES-9）。 GES酶家族与TEM和SHV超家族的区别最令人担忧的特征是它们明显能够进化成弱碳青霉烯酶，即能够水解碳青霉烯类抗生素的酶。 我们在2007年确定了GES-1的结构，最近结晶并确定了GES家族的另外两个成员GES-2和GES-5的结构，这两个成员都是GES-1酶的单点突变体，并且对碳青霉烯类抗生素亚胺培南显示出升高的活性。 此外，我们还确定了GES-5的脱酰缺陷突变体（E166 N）的结构。第二种酶Oih-1被分离出来

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. Beta-lactams are the most common antibiotics in clinical use and represent more than 60% of total world consumption of antimicrobial drugs. They include penicillins, cephalosporins, monobactams, penems and carbapenems, and over 50 antibiotics of this class are available on the market. A major mechanism of bacterial resistance to beta-lactam antibiotics is the production of beta-lactamases, enzymes that hydrolyze the conserved four-membered ring of beta-lactams in a two-step process. Firstly the beta-lactam ring is opened and the adduct binds covalently to an active site serine residue (the acylation step). This adduct is then deacylated by a water molecule held in place by a highly conserved glumate residue (Glu166), releasing the inactive peniciloic acid derivative of the drug. We are currently working on two recently-discovered beta-lactamase enzymes. The first, named GES-1 (Guiana Extended-Spectrum, after the country where it was first isolated) was initially described in 2000. This extended spectrum beta-lactamase (ESBL) is very distantly related to other class A beta-lactamases and produces resistance to penicillins and first-, second-, and some third-generation cephalosporins (e.g. ceftazidime) but not to monobactams and carbapenems. Since 2000, nine GES-type enzymes (GES-1 - GES-9) from different geographical locations have been described. The most alarming characteristic of the GES family of enzymes that distinguish them from the TEM and SHV superfamilies, is their apparent ability to evolve into weak carbapenemases, enzymes capable of hydrolyzing carbapenem antibiotics. We determined the GES-1 structure in 2007 and have recently crystallized and determined the structures of two other members of the GES family, GES-2 and GES-5, both of which a single point mutants of the GES-1 enzyme and which show elevated activity against the carbepenem antibiotic imipenem. In addition, we have also determined the structure of a deacylation deficient mutant (E166N) of GES-5. The second enzyme, Oih-1 was isolated