ATP Binding as a Key Target for Control of the Chemotaxis Kinase.

In bacterial chemotaxis, chemoreceptors in signaling complexes modulate the activity of two-component histidine kinase CheA in response to chemical stimuli. CheA catalyzes phosphoryl transfer from ATP to a histidinyl residue of its P1 domain. That phosphoryl group is transferred to two response regulators. Receptor control is almost exclusively at autophosphorylation, but the aspect of enzyme action on which that control acts is unclear. We investigated this by a kinetic analysis of activated kinase in signaling complexes. We found that phosphoryl transfer from ATP to P1 is an ordered sequential reaction in which the binding of ATP to CheA is the necessary first step; the second substrate, the CheA P1 domain, binds only to an ATP-occupied enzyme; and phosphorylated P1 is released prior to the second product, namely, ADP. We confirmed the crucial features of this kinetically deduced ordered mechanism by assaying P1 binding to the enzyme. In the absence of a bound nucleotide, there was no physiologically significant binding, but the enzyme occupied with a nonhydrolyzable ATP analog bound P1. Previous structural and computational analyses indicated that ATP binding creates the P1-binding site by ordering the "ATP lid." This process identifies the structural basis for the ordered kinetic mechanism. Recent mathematical modeling of kinetic data identified ATP binding as a focus of receptor-mediated kinase control. The ordered kinetic mechanism provides the biochemical logic of that control. We conclude that chemoreceptors modulate kinase by controlling ATP binding. Structural similarities among two-component kinases, particularly the ATP lid, suggest that ordered mechanisms and control of ATP binding are general features of two-component signaling.IMPORTANCE Our work provides important new insights into the action of the chemotaxis signaling kinase CheA by identifying the kinetic mechanism of its autophosphorylation as an ordered sequential reaction, in which the required first step is binding of ATP. These insights provide a framework for integrating previous kinetic, mathematical modeling, structural, simulation, and docking observations to conclude that chemoreceptors control the activity of the chemotaxis kinase by regulating binding of the autophosphorylation substrate ATP. Previously observed conformational changes in the ATP lid of the enzyme active site provide a structural basis for the ordered mechanism. Such lids are characteristic of two-component histidine kinases in general, suggesting that ordered sequential mechanisms and regulation by controlling ATP binding are common features of these kinases.

Structural and biochemical characterization of iminodiacetate oxidase from Chelativorans sp. BNC1.

Ethylenediaminetetraacetate (EDTA) is the most abundant organic pollutant in surface water because of its extensive usage and the recalcitrance of stable metal-EDTA complexes. A few bacteria including Chelativorans sp. BNC1 can degrade EDTA with a monooxygenase to ethylenediaminediacetate (EDDA) and then use iminodiacetate oxidase (IdaA) to further degrade EDDA into ethylenediamine in a two-step oxidation. To alleviate EDTA pollution into the environment, deciphering the mechanisms of the metabolizing enzymes is an imperative prerequisite for informed EDTA bioremediation. Although IdaA cannot oxidize glycine, the crystal structure of IdaA shows its tertiary and quaternary structures similar to those of glycine oxidases. All confirmed substrates, EDDA, ethylenediaminemonoacetate, iminodiacetate and sarcosine are secondary amines with at least one N-acetyl group. Each substrate was bound at the re-side face of the isoalloxazine ring in a solvent-connected cavity. The carboxyl group of the substrate was bound by Arg265 and Arg307 . The catalytic residue, Tyr250 , is under the hydrogen bond network to facilitate its deprotonation acting as a general base, removing an acetate group of secondary amines as glyoxylate. Thus, IdaA is a secondary amine oxidase, and our findings improve understanding of molecular mechanism involved in the bioremediation of EDTA and the metabolism of secondary amines.

Biochemical and Structural Analysis of Substrate Specificity of a Phenylalanine Ammonia-Lyase.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from l-phenylalanine to give trans-cinnamate. In monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of p-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (Sorghum bicolor; SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared with that of dicotyledonous plants. The side chain of histidine-123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from 189Ala-Ser-Gly191 residues and the bound l-phenylalanine and l-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E2 reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase.

Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2 -dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA(2-) into EmoA identified an intricate hydrogen bond network connected to Tyr(71) , which should potentially lower its pKa. Tyr(71) , along with nearby Glu(70) and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channelling of FMNH2 .

Dissociation of MIF-rpS3 complex and sequential NF-κB activation is involved in IR-induced metastatic conversion of NSCLC.

Frequent relapse and spreading of tumors during radiotherapy are principal obstacles to treatment of non-small cell lung cancer (NSCLC). In this study, we aimed to investigate how macrophage migration inhibitory factor (MIF) which is expressed at high levels in metastatic and primary lung cancer cells could regulate NSCLC metastasis in response to ionizing radiation (IR). The results indicated that MIF and ribosomal protein S3 (rpS3) were shown to be connected to inflammation, proliferation, and metastasis of NSCLC via IR-induced activation of the NF-κB pathway. Under unirradiated conditions, MIF physically established a complex with rpS3. MIF-rpS3 dissociation induced by IR activated NF-κB and made the expression of target genes of this factor transactivated in two NSCLC cell lines, A549, and NCI-H358. We also found that IR-induced dissociation of this complex led to increased secretion of pro-inflammatory cytokines and modulated the expression of epithelial-mesenchymal transition marker proteins. Finally, the effects of IR-induced dissociation of the MIF-rpS3 complex on tumor metastasis were confirmed by in vivo xenograft studies. Taken together, the present study revealed that dissociation of the MIF-rpS3 complex and subsequent activation of NF-κB is a critical post-IR exposure event that accounts for IR-induced metastatic conversion of NSCLC.

Inhibitory effect of traditional oriental medicine-derived monoamine oxidase B inhibitor on radioresistance of non-small cell lung cancer.

Increased survival of cancer cells mediated by high levels of ionizing radiation (IR) reduces the effectiveness of radiation therapy for non-small cell lung cancer (NSCLC). In the present study, danshensu which is a selected component of traditional oriental medicine (TOM) compound was found to reduce the radioresistance of NSCLC by inhibiting the nuclear factor-κB (NF-κB) pathway. Of the various TOM compounds reported to inhibit the IR activation of NF-κB, danshensu was chosen as a final candidate based on the results of structural comparisons with human metabolites and monoamine oxidase B (MAOB) was identified as the putative target enzyme. Danshensu decreased the activation of NF-κB by inhibiting MAOB activity in A549 and NCI-H1299 NSCLC cells. Moreover, it suppressed IR-induced epithelial-to-mesenchymal transition, expressions of NF-κB-regulated prosurvival and proinflammatory genes, and in vivo radioresistance of mouse xenograft models. Taken together, this study shows that danshensu significantly reduces MAOB activity and attenuates NF-κB signaling to elicit the radiosensitization of NSCLC.

Suppression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice by transduced Tat-Annexin protein.

We examined that the protective effects of ANX1 on 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced skin inflammation in animal models using a Tat-ANX1 protein. Topical application of the Tat-ANX1 protein markedly inhibited TPAinduced ear edema and expression levels of cyclooxygenase-2 (COX-2) as well as pro-inflammatory cytokines such as interleukin- 1 beta (IL-1 ß), IL-6, and tumor necrosis factor-alpha (TNF-α). Also, application of Tat-ANX1 protein significantly inhibited nuclear translocation of nuclear factor-kappa B (NF-κ B) and phosphorylation of p38 and extracellular signalregulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TPA-treated mice ears. The results indicate that Tat-ANX1 protein inhibits the inflammatory response by blocking NF-κ B and MAPK activation in TPA-induced mice ears. Therefore, the Tat-ANX1 protein may be useful as a therapeutic agent against inflammatory skin diseases.

Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis.

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.