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1.
Protein Expr Purif ; 188: 105964, 2021 12.
Article in English | MEDLINE | ID: mdl-34454050

ABSTRACT

The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, Km and Vmax were 11.2 µM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments.


Subject(s)
Aeromonadaceae/enzymology , Bacterial Proteins/genetics , Catechol 1,2-Dioxygenase/genetics , Catechols/metabolism , Aeromonadaceae/chemistry , Aeromonadaceae/classification , Aeromonadaceae/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Catechol 1,2-Dioxygenase/chemistry , Catechol 1,2-Dioxygenase/isolation & purification , Catechol 1,2-Dioxygenase/metabolism , Catechols/chemistry , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Phylogeny , Protein Multimerization , Pyrogallol/chemistry , Pyrogallol/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
2.
PLoS One ; 15(9): e0233823, 2020.
Article in English | MEDLINE | ID: mdl-32941430

ABSTRACT

Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log2 fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions.


Subject(s)
Aeromonadaceae/metabolism , Iron/metabolism , Lignin/metabolism , Aeromonadaceae/enzymology , Aeromonadaceae/growth & development , Bacterial Proteins/metabolism , Biomass , Oxidation-Reduction , Sulfatases/metabolism
3.
J Microbiol ; 54(2): 114-21, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26832667

ABSTRACT

The gene product of dddC (Uniprot code G5CZI2), from the Gram-negative marine bacterium Oceanimonas doudoroffii, is a methylmalonate-semialdehyde dehydrogenase (OdoMMSDH) enzyme. MMSDH is a member of the aldehyde dehydrogenase superfamily, and it catalyzes the NAD-dependent decarboxylation of methylmalonate semialdehyde to propionyl-CoA. We determined the crystal structure of OdoMMSDH at 2.9 Å resolution. Among the twelve molecules in the asymmetric unit, six subunits complexed with NAD, which was carried along the protein purification steps. OdoMMSDH exists as a stable homodimer in solution; each subunit consists of three distinct domains: an NAD-binding domain, a catalytic domain, and an oligomerization domain. Computational modeling studies of the OdoMMSDH structure revealed key residues important for substrate recognition and tetrahedral intermediate stabilization. Two basic residues (Arg103 and Arg279) and six hydrophobic residues (Phe150, Met153, Val154, Trp157, Met281, and Phe449) were found to be important for tetrahedral intermediate binding. Modeling data also suggested that the backbone amide of Cys280 and the side chain amine of Asn149 function as the oxyanion hole during the enzymatic reaction. Our results provide useful insights into the substrate recognition site residues and catalytic mechanism of OdoMMSDH.


Subject(s)
Aeromonadaceae/enzymology , Methylmalonate-Semialdehyde Dehydrogenase (Acylating)/chemistry , Crystallography, X-Ray , Models, Molecular , NAD/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary
4.
J Protein Chem ; 22(6): 515-9, 2003 Aug.
Article in English | MEDLINE | ID: mdl-14703984

ABSTRACT

Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3'(2')-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in V(max) by a factor of 1.1 x 10(4). Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.


Subject(s)
Aeromonadaceae/enzymology , Metals/metabolism , Mutagenesis/genetics , Phosphoenolpyruvate Carboxykinase (ATP)/genetics , Phosphoenolpyruvate Carboxykinase (ATP)/metabolism , Aeromonadaceae/genetics , Binding Sites , Circular Dichroism , Computer Simulation , Kinetics , Models, Molecular , Molecular Conformation , Mutation, Missense/genetics , Phosphoenolpyruvate Carboxykinase (ATP)/chemistry , Protein Structure, Tertiary
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