Ha Il Jung

Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C β-lactamase

The emergence and dissemination of extended‐spectrum (ES) β‐lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by third‐generation β‐lactam antibiotics like ceftazidime. CMY‐10 is a plasmid‐encoded class C β‐lactamase with a wide spectrum of substrates. Unlike the well‐studied class C ES β‐lactamase from Enterobacter cloacae GC1, the Ω‐loop does not affect the active site conformation and the catalytic activity of CMY‐10. Instead, a three‐amino‐acid deletion in the R2‐loop appears to be responsible for the ES activity of CMY‐10. According to the crystal structure solved at 1.55 Å resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side‐chains of β‐lactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY‐10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 β‐lactamase reveal that the introduction of deletion mutations into the R2‐loop is able to extend the substrate spectrum of class C non‐ES β‐lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2‐loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2‐loop can be considered as an operative molecular strategy of class C β‐lactamases to extend their substrate spectrum.

Analyses of Mlc–IIBGlc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration

In Escherichia coli, glucose-dependent transcriptional induction of genes encoding a variety of sugar-metabolizing enzymes and transport systems is mediated by the phosphorylation state-dependent interaction of membrane-bound enzyme IICBGlc (EIICBGlc) with the global repressor Mlc. Here we report the crystal structure of a tetrameric Mlc in a complex with four molecules of enzyme IIBGlc (EIIB), the cytoplasmic domain of EIICBGlc. Each monomer of Mlc has one bound EIIB molecule, indicating the 1:1 stoichiometry. The detailed view of the interface, along with the high-resolution structure of EIIB containing a sulfate ion at the phosphorylation site, suggests that the phosphorylation-induced steric hindrance and disturbance of polar intermolecular interactions impede complex formation. Furthermore, we reveal that Mlc possesses a built-in flexibility for the structural adaptation to its target DNA and that interaction of Mlc with EIIB fused only to dimeric proteins resulted in the loss of its DNA binding ability, suggesting that flexibility of the Mlc structure is indispensable for its DNA binding.

Dissemination of SHV-12 and Characterization of New AmpC-Type Beta-Lactamase Genes among Clinical Isolates of Enterobacter Species in Korea

ABSTRACT To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of Enterobacter species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the ampC genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four ampC genes (blaEcloK992004.1, blaEcloK995120.1, blaEcloK99230, and blaEareK9911729) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal ampC gene (blaEcloMHN1) of Enterobacter cloacae MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two ampC genes (blaEcloK9973 and blaEcloK9914325) showed close similarity to the chromosomal ampC gene (blaEcloQ908R) of E. cloacae Q908R (99.7% identity). The results from phylogenetic analysis suggested that six ampC genes could originate from blaEcloMHN1 or blaEcloQ908R.

Investigation of extended-spectrum β-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

Aims:  Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended‐spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended‐spectrum β‐lactamases (ESBLs).

Crystal Structure of Filamentous Aggregates of Human DJ-1 Formed in an Inorganic Phosphate-dependent Manner

Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-Å crystal structure, DJ-1 dimers are linearly stacked through Pi-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.

Crystallization and preliminary X-ray crystallographic analyses of CMY-1 and CMY-10, plasmidic class C β-lactamases with extended substrate spectrum

Plasmid-encoded class C beta-lactamases, including CMY-1 and CMY-10, hydrolyze the lactam bonds of beta-lactam antibiotics, inducing therapeutic failure and a lack of eradication of clinical isolates by third-generation cephalosporins or cephamycins. Therefore, the enzymes are potential targets for developing agents against pathogens isolated from patients suffering from wound infection, urinary tract infection or pneumonia. CMY-1 and CMY-10 were purified and crystallized at 298 K. X-ray diffraction data from CMY-1 and CMY-10 crystals have been collected to 2.5 and 1.5 A resolution, respectively, using synchrotron radiation. The crystals of the two proteins are isomorphous and belong to the primitive monoclinic space group P2(1).

Crystallization and Preliminary X-Ray Crystallographic Analysis of CTXM- 15, an Extended-spectrum β-Lactamase Conferring Worldwide Emerging Antibiotic Resistance

CTX-M-15, an extended-spectrum β-lactamase emerging worldwide, hydrolyzes lactam ring of β-lactam antibiotics, and thus causes therapeutic failure and a lack of eradication of pathogenic bacteria by third-generation β-lactams. Therefore, the enzyme is a potential target for developing agents against pathogens isolated from patients suffering from nosocomial infections. The CTX-M-15 protein was purified and crystallized at 298 K. X-ray diffraction data from CTX-M-15 crystal have been collected to 1.46 Å resolution using synchrotron radiation. The crystal of CTX-M-15 belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.50, b = 44.23, and c = 116.92 Å. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 41.26%.

Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum β-lactamase conferring severe antibiotic resistance

OXA-17, an extended-spectrum β-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates β-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino β-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 Å resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P212121, with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 Å. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%.

Characterization and molecular epidemiology of Enterobacter cloacae clinical isolates producing extended-spectrum β-lactamases

Seok Hoon Jeong, Il Kwon Bae, Seung Ghyu Sohn, Ha Il Jung, Young Jun An, Eui Suk Sohn, Jung Hun Lee, and Sang Hee Lee 1 Department of Laboratory Medicine and Research Institute for Antimicrobial Resistance, Kosin University College of Medicine, Busan, 602-030, Republic of Korea 2 Department of Biological Sciences, School of Biotechnology and Environmental Engineering, Myongji University, San 38-2 Namdong, Yongin, Gyeonggido, 449-728, Republic of Korea

Crystallization and preliminary X-ray crystallographic analysis of a novel histidinol-phosphate phosphatase from Thermococcus onnurineus NA1.

The TON_0887 gene product from Thermococcus onnurineus NA1 is a 240-residue protein that has histidinol-phosphate phosphatase (HolPase) activity. According to analysis of its primary structure, the TON_0887 gene product is a monofunctional HolPase that belongs to the DDDD superfamily. This contrasts with the generally accepted classification that bifunctional HolPases belong to the DDDD superfamily. The TON_0887 gene product was purified and crystallized at 295 K. A 2.2 A resolution data set was collected using synchrotron radiation. The TON-HolPase crystals belonged to space group P222(1), with unit-cell parameters a = 40.88, b = 46.89, c = 148.03 A. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 48.3%.