窄食单胞菌属。在整个环境中发现了对许多β-内酰胺抗生素的固有抗性。在寡养单胞菌属中鉴定的CESS-1。KCTC12332是一种未表征的A类β-内酰胺酶。这里,CESS-1显示出对青霉素(青霉素G和氨苄西林)和头孢菌素(头孢氨苄,头孢克洛,和头孢噻肟),而其对碳青霉烯类(亚胺培南和美罗培南)的活性可忽略不计。虽然头孢克洛,头孢氨苄,和氨苄青霉素具有相似的结构,具有相同的R1侧链,CESS-1对三种β-内酰胺抗生素的催化参数不同。cephaclor的kcat值,头孢氨苄,和氨苄青霉素分别为1249.6s-1、204.3s-1和69.8s-1,伴随的KM值为287.6μM,236.7μM,和28.8μM,分别。值得注意的是,CESS-1区分头孢克洛和头孢氨苄,只有一个结构差异:C3的-Cl(头孢克洛)和-CH3(头孢氨苄)。根据头孢克洛酰化的CESS-1的三个E166Q突变体的结构比较,头孢氨苄,和氨苄青霉素,底物R1侧链及其接触β5-β6环的协同位置变化会影响酰基酶中间状态下Asn170与脱酰水之间的距离。这与CESS-1对三种结构相似的β-内酰胺抗生素的差异水解活性直接相关。
OBJECTIVE: Stenotrophomonas spp. intrinsically resistant to many β-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A β-lactamase. The goal of this study was to reveal biochemical and structural characteristics of CESS-1.
METHODS: The hydrolytic activities of CESS-1 towards penicillins (penicillin G and ampicillin), cephalosporins (cephalexin, cefaclor, and cefotaxime), and carbapenems (imipenem and meropenem) was spectrophotometrically monitored. Structural information on E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin were determined by X-ray crystallography.
RESULTS: CESS-1 displayed hydrolytic activities toward penicillins and cephalosporins, with negligible activity toward carbapenems. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward them were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were 1249.6 s-1, 204.3 s-1, and 69.8 s-1, respectively, with the accompanying KM values of 287.6 μM, 236.7 μM, and 28.8 μM, respectively.
CONCLUSIONS: CESS-1 was able to discriminate between cefaclor and cephalexin with a single structural difference at C3 position: -Cl (cefaclor) and -CH3 (cephalexin). Structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin, revealed that cooperative positional changes in the R1 side chain of substrates and their interaction with the β5-β6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar β-lactam antibiotics.