Myoung-Ki Hong

Crystal Structure of Malonyl CoA-Acyl Carrier Protein Transacylase from Xanthomanous oryzae pv. oryzae and Its Proposed Binding with ACP

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.

Structure of ginseng major latex-like protein 151 and its proposed lysophosphatidic acid-binding mechanism.

Lysophosphatidic acid (LPA) is a phospholipid growth factor with myriad effects on biological systems. LPA is usually present bound to animal plasma proteins such as albumin or gelsolin. When LPA complexes with plasma proteins, it binds to its cognate receptors with higher affinity than when it is free. Recently, gintonin from ginseng was found to bind to LPA and to activate mammalian LPA receptors. Gintonin contains two components: ginseng major latex-like protein 151 (GLP) and ginseng ribonuclease-like storage protein. Here, the crystal structure of GLP is reported, which belongs to the plant Bet v 1 superfamily, and a model is proposed for how GLP binds LPA. Amino-acid residues of GLP recognizing LPA were identified using site-directed mutagenesis and isothermal titration calorimetry. The resulting GLP mutants were used to study the activation of LPA receptor-dependent signalling pathways. In contrast to wild-type GLP, the H147A mutant did not bind LPA, elicit intracellular Ca(2+) transients in neuronal cells or activate Ca(2+)-dependent Cl(-) channels in Xenopus oocytes. Based on these results, a mechanism by which GLP recognizes LPA and its requirement to activate G protein-coupled LPA receptors to elicit diverse biological responses were proposed.

Crystal structure of cytochrome P450 CYP105N1 from Streptomyces coelicolor, an oxidase in the coelibactin siderophore biosynthetic pathway

The genome sequence of Streptomyces coelicolor contains 18 cytochrome P450 enzymes. The recombinant CYP105N1 protein has been expressed in Escherichia coli and purified, and we report the biochemical and structural characterization of CYP105N1 from S. coelicolor. The purified protein exhibited the typical CO-binding spectrum of P450 enzymes and type I binding spectra with estradiol and a coelibactin analog. The oxidation of estradiol by CYP105N1, supported by H(2)O(2), produced estriol. The crystal structure of CYP105N1 was determined at 2.9 Å resolution. An unexpected wide open binding pocket located above the heme group was identified, with a volume of approximately 4299 Å(3). These results suggest that the large open pocket to the active site may be a key feature for easy access of the peptidyl carrier protein-bound substrate to perform the hydroxylation reaction. A molecular docking model with coelibactin showed that the phenyl group of coelibactin is located <4 Å away from the heme-iron, suggesting that CYP105N1 may be involved in the hydroxylation of the phenyl ring of the coelibactin precursor during biosynthesis.

Structure of ADC‐68, a novel carbapenem‐hydrolyzing class C extended‐spectrum β‐lactamase isolated from Acinetobacter baumannii

Outbreaks of multidrug-resistant bacterial infections have become more frequent worldwide owing to the emergence of several different classes of β-lactamases. In this study, the molecular, biochemical and structural characteristics of an Acinetobacter-derived cephalosporinase (ADC)-type class C β-lactamase, ADC-68, isolated from the carbapenem-resistant A. baumannii D015 were investigated. The blaADC-68 gene which encodes ADC-68 was confirmed to exist on the chromosome via Southern blot analysis and draft genome sequencing. The catalytic kinetics of β-lactams and their MICs (minimum inhibitory concentrations) for A. baumannii D015 and purified ADC-68 (a carbapenemase obtained from this strain) were assessed: the strain was resistant to penicillins, narrow-spectrum and extended-spectrum cephalosporins, and carbapenems, which were hydrolyzed by ADC-68. The crystal structure of ADC-68 was determined at a resolution of 1.8 Å. The structure of ADC-68 was compared with that of ADC-1 (a non-carbapenemase); differences were found in the central part of the Ω-loop and the C-loop constituting the edge of the R1 and R2 subsites and are close to the catalytic serine residue Ser66. The ADC-68 C-loop was stabilized in the open conformation of the upper R2 subsite and could better accommodate carbapenems with larger R2 side chains. Furthermore, a wide-open conformation of the R2-loop allowed ADC-68 to bind to and hydrolyze extended-spectrum cephalosporins. Therefore, ADC-68 had enhanced catalytic efficiency against these clinically important β-lactams (extended-spectrum cephalosporins and carbapenems). ADC-68 is the first reported enzyme among the chromosomal class C β-lactamases to possess class C extended-spectrum β-lactamase and carbapenemase activities.

Structure-based studies on the metal binding of two-metal-dependent sugar isomerases

Two‐metal‐dependent sugar isomerases are important in the synthesis of rare sugars. Many of their properties, specifically their metal dependency, have not been sufficiently explored. Here we used X‐ray crystallography, site‐directed mutagenesis, isothermal titration calorimetry and electron paramagnetic resonance spectroscopy to investigate the molecular determinants of the metal‐binding affinity of l‐rhamnose isomerase, a two‐Mn2+‐dependent isomerase from Bacillus halodurans (BHRI). The crystal structure of BHRI confirmed the presence of two metal ion‐binding sites: a structural metal ion‐binding site for substrate binding, and a catalytic metal ion‐binding site that catalyzes a hydride shift. One conserved amino acid, W38, in wild‐type BHRI was identified as a critical residue for structural Mn2+ binding and thus the catalytic efficiency of BHRI. This function of W38 was explored by replacing it with other amino acids. Substitution by Phe, His, Lys, Ile or Ala caused complete loss of catalytic activity. The role of W38 was further examined by analyzing the crystal structure of wild‐type BHRI and two inactive mutants of BHRI (W38F and W38A) in complex with Mn2+. A structural comparison of the mutants and the wild‐type revealed differences in their coordination of Mn2+, including changes in metal–ligand bond length and affinity for Mn2+. The role of W38 was further confirmed in another two‐metal‐dependent enzyme: xylose isomerase from Bacillus licheniformis. These data suggest that W38 stabilizes protein–metal complexes and in turn assists ligand binding during catalysis in two‐metal‐dependent isomerases.

Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) from Escherichia coli

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.

The crystal structure of the d-alanine-d-alanine ligase from Acinetobacter baumannii suggests a flexible conformational change in the central domain before nucleotide binding

Acinetobacter baumannii, which is emerging as a multidrugresistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A. baumannii (AbDDL) at a resolution of 2.2 Å. The asymmetric unit contained six protomers of AbDDL. Five protomers had a closed conformation in the central domain, while one protomer had an open conformation in the central domain. The central domain with an open conformation did not interact with crystallographic symmetry-related protomers and the conformational change of the central domain was not due to crystal packing. The central domain of AbDDL can have an ensemble of the open and closed conformations before the binding of substrate ATP. The conformational change of the central domain is important for the catalytic activity and the detail information will be useful for the development of inhibitors against AbDDL and putative antibacterial agents against A. baumannii. The AbDDL structure was compared with that of other DDLs that were in complex with potent inhibitors and the catalytic activity of AbDDL was confirmed using enzyme kinetics assays.

A physical association between the human mutY homolog (hMYH) and DNA topoisomerase II-binding protein 1 (hTopBP1) regulates Chk1-induced cell cycle arrest in HEK293 cells

BackgroundHuman DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is.ResultsWe documented the interaction between hTopBP1 and hMYH and showed that this interaction increased in a hydroxyurea-dependent manner. We also mapped the hMYH-interacting region of hTopBP1 (residues 444–991). In addition, we investigated several cell cycle-related proteins and found that co-knockdown of hTopBP1 and hMYH significantly diminished cell cycle arrest due to compromised checkpoint kinase 1 (Chk1) activation. Moreover, we observed that hMYH was essential for the accumulation of hTopBP1 on damaged DNA, where hTopBP1 interacts with hRad9, a component of the Rad9-Hus1-Rad1 complex. The accumulation of hTopBP1 on chromatin and its subsequent interaction with hRad9 lead to cell cycle arrest, a process mediated by Chk1 phosphorylation and ataxia telangiectasia and Rad3-related protein (ATR) activation.ConclusionsOur results suggested that hMYH is necessary for the accumulation of hTopBP1 to DNA damage lesion to induce the association of hTopBP1 with 9-1-1 and that the interaction between hMYH and hTopBP1 is essential for Chk1 activation. Therefore, we suggest that the interaction between hMYH and hTopBP1 is crucial for activation of the ATR-mediated cell cycle checkpoint.

Expression, crystallization and preliminary X-ray crystallographic analysis of cellobiose 2-epimerase from Dictyoglomus turgidum DSM 6724

Cellobiose 2-epimerase epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=63.9, b=85.1, c=79.8 Å, β=110.8°. With a Matthews coefficient VM of 2.18 Å3 Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=55.9, b=80.0, c=93.7 Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26 Å3 Da(-1) and a solvent content of 45.6%.

Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.