Characterization of the extended substrate spectrum of the class A ß-lactamase CESS-1 from Stenotrophomonas sp. and structure-based investigation into its substrate preference.

Stenotrophomonas spp. intrinsically resistant to many ß-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A ß-lactamase. Here, CESS-1 was revealed to display hydrolytic activities toward penicillins (penicillin G and ampicillin) and cephalosporins (cephalexin, cefaclor, and cefotaxime), while its activity toward carbapenems (imipenem and meropenem) was negligible. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward the three ß-lactam antibiotics were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were calculated to be 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. Remarkably, CESS-1 discriminates cefaclor and cephalexin with only one structural difference: -Cl (cefaclor) and -CH3 (cephalexin) at C3. According to structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, and ampicillin, the cooperative positional changes of the R1 side chain of substrates and its contacting ß5-ß6 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 ß-lactam antibiotics.

Control of the signaling role of PtdIns(4)P at the plasma membrane through H2O2-dependent inactivation of synaptojanin 2 during endocytosis.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is implicated in various processes, including hormone-induced signal transduction, endocytosis, and exocytosis in the plasma membrane. However, how H2O2 accumulation regulates the levels of PtdIns(4,5)P2 in the plasma membrane in cells stimulated with epidermal growth factors (EGFs) is not known. We show that a plasma membrane PtdIns(4,5)P2-degrading enzyme, synaptojanin (Synj) phosphatase, is inactivated through oxidation by H2O2. Intriguingly, H2O2 inhibits the 4-phosphatase activity of Synj but not the 5-phosphatase activity. In EGF-activated cells, the oxidation of Synj dual phosphatase is required for the transient increase in the plasma membrane levels of phosphatidylinositol 4-phosphate [PtdIns(4)P], which can control EGF receptor-mediated endocytosis. These results indicate that intracellular H2O2 molecules act as signaling mediators to fine-tune endocytosis by controlling the stability of plasma membrane PtdIns(4)P, an intermediate product of Synj phosphoinositide dual phosphatase.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

Structural and functional identification of the uncharacterized metallo-ß-lactamase superfamily protein TW9814 as a phosphodiesterase with unique metal coordination.

Metallo-ß-lactamase (MBL) superfamily proteins have a common αß/ßα sandwich fold and perform a variety of functions through metal-mediated catalysis. However, because of the enormous scale of this superfamily, only a small percentage of the proteins belonging to the superfamily have been annotated structurally or functionally to date. Therefore, much remains unknown about the MBL superfamily proteins. Here, TW9814, a hypothetical MBL superfamily protein, was structurally and functionally investigated. Guided by the crystal structure of dimeric TW9814, it was demonstrated that TW9814 functions as a phosphodiesterase (PDE) in the presence of divalent metal ions such as manganese(II) or nickel(II). A docking model between TW9814 and the substrate bis(p-nitrophenyl)phosphate (bpNPP) showed the importance of the dimerization of TW9814 for its bpNPP-hydrolyzing activity and for the interaction between the enzyme and the substrate. TW9814 showed outstanding catalytic efficiency (kcat/Km) under alkaline conditions compared with other PDEs. The activity of TW9814 appears to be regulated through a disulfide bond, which is a feature that is not present in other MBL superfamily members. This study provides a platform for the functional characterization of other hypothetical proteins of the MBL or other superfamilies.

Crystal structure of AmpC BER and molecular docking lead to the discovery of broad inhibition activities of halisulfates against ß-lactamases.

AmpC BER is an extended-spectrum (ES) class C ß-lactamase with a two-amino-acid insertion in the H10 helix region located at the boundary of the active site compared with its narrow spectrum progenitor. The crystal structure of the wild-type AmpC BER revealed that the insertion widens the active site by restructuring the flexible H10 helix region, which is the structural basis for its ES activity. Besides, two sulfates originated from the crystallization solution were observed in the active site. The presence of sulfate-binding subsites, together with the recognition of ring-structured chemical scaffolds by AmpC BER, led us to perform in silico molecular docking experiments with halisulfates, natural products isolated from marine sponge. Inspired by the snug fit of halisulfates within the active site, we demonstrated that halisulfate 3 and 5 significantly inhibit ES class C ß-lactamases. Especially, halisulfate 5 is comparable to avibactam in terms of inhibition efficiency; it inhibits the nitrocefin-hydrolyzing activity of AmpC BER with a Ki value of 5.87 µM in a competitive manner. Furthermore, halisulfate 5 displayed moderate and weak inhibition activities against class A and class B/D enzymes, respectively. The treatment of ß-lactamase inhibitors (BLIs) in combination with ß-lactam antibiotics is a working strategy to cope with infections by pathogens producing ES ß-lactamases. Considering the emergence and dissemination of enzymes insensitive to clinically-used BLIs, the broad inhibition spectrum and structural difference of halisulfates would be used to develop novel BLIs that can escape the bacterial resistance mechanism mediated by ß-lactamases.

Novel inhibition mechanism of carbapenems on the ACC-1 class C ß-lactamase.

The hydrolysis of ß-lactam antibiotics by class C ß-lactamases proceeds through the acylation and the rate-determining deacylation steps mediated by the nucleophilic serine and the deacylation water, respectively. The pose of poor substrates such as carbapenems in the acylated enzyme is responsible for the low efficient deacylation reaction. Here we present the crystal structures of the Y150F variant of the ACC-1 class C ß-lactamase in the apo and acylated states. In the acylated enzyme complexed with two carbapenems, imipenem and meropenem, the lactam carbonyl oxygen is located in the oxyanion hole. However, the five-membered pyrroline ring displays a novel orientation that has not been reported so far. The ring is rotated such that its C3 carboxylate makes salt bridges with Lys67 and Ly315, which is accompanied by the side-chain rotamer change of Phe150. The C3 carboxylate is placed where the deacylation water occupies in the apo-enzyme, which, together with the displacement of the catalytic base residue at position 150, explains why carbapenems are poor substrates of ACC-1.

In vitro and in vivo Inhibitory Activity of NADPH Against the AmpC BER Class C ß-Lactamase.

ß-Lactamase-mediated resistance to ß-lactam antibiotics has been significantly threatening the efficacy of these clinically important antibacterial drugs. Although some ß-lactamase inhibitors are prescribed in combination with ß-lactam antibiotics to overcome this resistance, the emergence of enzymes resistant to current inhibitors necessitates the development of novel ß-lactamase inhibitors. In this study, we evaluated the inhibitory effect of dinucleotides on an extended-spectrum class C ß-lactamase, AmpC BER. Of the dinucleotides tested, NADPH, a cellular metabolite, decreased the nitrocefin-hydrolyzing activity of the enzyme with a Ki value of 103 µM in a non-covalent competitive manner. In addition, the dissociation constant (KD) between AmpC BER and NADPH was measured to be 40 µM. According to our in vitro susceptibility study based on growth curves, NADPH restored the antibacterial activity of ceftazidime against a ceftazidime-resistant Escherichia coli BER strain producing AmpC BER. Remarkably, a single dose of combinatory treatment with NADPH and ceftazidime conferred marked therapeutic efficacy (100% survival rate) in a mouse model infected by the E. coli BER strain although NADPH or ceftazidime alone failed to prevent the lethal bacterial infection. These results may offer the potential of the dinucleotide scaffold for the development of novel ß-lactamase inhibitors.