ABSTRACT
Xanthomonas citri subsp. citri (Xac) is the causative agent of citrus canker, a plant disease that significantly impacts citriculture. In earlier work, we showed that alkylated derivatives of gallic acid have antibacterial action against Xac and target both the cell division protein FtsZ and membrane integrity in Bacillus subtilis. Here, we have purified native XacFtsZ and characterized its GTP hydrolysis and polymerization properties. In a surprising manner, inhibition of XacFtsZ activity by alkyl gallates is not as strong as observed earlier with B. subtilis FtsZ. As the alkyl gallates efficiently permeabilize Xac membranes, we propose that this is the primary mode of antibacterial action of these compounds.
Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cytoskeletal Proteins/isolation & purification , Cytoskeletal Proteins/metabolism , Xanthomonas/enzymology , Anti-Bacterial Agents/pharmacology , Cell Membrane/drug effects , Citrus/microbiology , Enzyme Inhibitors/metabolism , Gallic Acid/pharmacology , Guanosine Triphosphate/metabolism , Hydrolysis , Plant Diseases/microbiology , Protein Multimerization , Xanthomonas/drug effectsABSTRACT
Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single- and double-mixing stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.
Subject(s)
Bacterial Proteins/chemistry , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Rhodococcus/enzymology , Dinitrocresols/chemistry , Kinetics , Models, Chemical , Oxidation-Reduction , Putrescine/chemistryABSTRACT
A recently discovered class of bicovalent flavoproteins is an interesting group of enzymes because of their unusual cofactor binding mode, their open active sites and the bulky substrates they can accept. Through a sequence comparison study we have identified a conserved sequence region in bicovalent flavoproteins that is different from monocovalent flavoproteins. Based on this and the available structural information we have designed mutants of the prototype monocovalent flavoprotein, 6-hydroxy-d-nicotine oxidase (6HDNO), in order to introduce a second cofactor-protein linkage. Two amino acid replacements, namely histidine 130 to a cysteine and leucine 138 to a histidine, were sufficient to create a bicovalent 6HDNO. The introduced cysteine forms a covalent bond with FAD as found in natural bicovalent flavoproteins, while the second mutation was found to be essential to facilitate the formation of the cysteinyl linkage. This points to an important role of the introduced histidine in stabilizing a negative charge of the isoalloxazine ring during covalent flavinylation. The His130Cys/Leu138His 6HDNO is still active and shows a higher midpoint redox potential when compared to wild-type 6HDNO. This agrees well with the previous studies that have shown that bicovalent flavoenzymes have extremely high redox potentials.
Subject(s)
Flavoproteins/chemistry , Flavoproteins/genetics , Mutagenesis , Oxidoreductases/chemistry , Oxidoreductases/genetics , Flavoproteins/metabolism , Oxidoreductases/metabolism , Protein Conformation , Protein EngineeringABSTRACT
Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles the sequence-related human monoamine oxidases A and B. This similarity is striking in the flavin-binding site even if PuO does not covalently bind the cofactor as do the monoamine oxidases. A remarkable conserved feature is the cis peptide conformation of the Tyr residue whose conformation is important for substrate recognition in the active site cavity. The structure of PuO in complex with the reaction product reveals that Glu324 is crucial in recognizing the terminal amino group of the diamine substrate and explains the narrow substrate specificity of the enzyme. The structural analysis also provides clues for identification of residues that are responsible for the competitive binding of ADP versus FAD (~50% of wild-type PuO monomers isolated are occupied by ADP instead of FAD). By replacing Pro15, which is part of the dinucleotide-binding domain, enzyme preparations were obtained that are almost 100% in the FAD-bound form. Furthermore, mutants have been designed and prepared that form a covalent 8α-S-cysteinyl-FAD linkage. These data provide new insights into the molecular basis for substrate recognition in amine oxidases and demonstrate that engineering of flavoenzymes to introduce covalent linkage with the cofactor is a possible route to develop more stable protein molecules, better suited for biocatalytic purposes.
