ABSTRACT
We have found for the first time that a chromosomal gene, mlr6787, in Mesorhizobium loti encodes the pyridoxine degradative enzyme alpha-(N-acetylaminomethylene)succinic acid (AAMS) amidohydrolase. The recombinant enzyme expressed in Escherichia coli cells was homogeneously purified and characterized. The enzyme consisted of two subunits each with a molecular mass of 34,000+/-1,000 Da, and exhibited Km and kcat values of 53.7+/-6 microM and 307.3+/-12 min(-1), respectively. The enzyme required no cofactor or metal ion. The primary structure of AAMS amidohydrolase was elucidated for the first time here. The primary structure of the enzyme protein showed no significant identity to those of known hydrolase proteins and low homology to those of fluoroacetate dehalogenase (PDB code, 1Y37), haloalkane dehalogenase (1K5P), and aryl esterase (1VA4).
Subject(s)
Alphaproteobacteria/enzymology , Amidohydrolases/genetics , Gene Expression/genetics , Hydrolases/genetics , Pyridoxine/metabolism , Succinate-Semialdehyde Dehydrogenase/isolation & purification , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Bacterial Proteins/genetics , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Escherichia coli/genetics , Genes, Bacterial/genetics , Hydrogen-Ion Concentration , Pyridoxine/chemistry , Pyridoxine/genetics , Succinate-Semialdehyde Dehydrogenase/chemistry , Succinate-Semialdehyde Dehydrogenase/genetics , Temperature , gamma-Aminobutyric Acid/analogs & derivatives , gamma-Aminobutyric Acid/chemistry , gamma-Aminobutyric Acid/geneticsABSTRACT
The gene (mlr6788) of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 has been identified as a gene coding for 2-methyl-3-hydroxypyridine-5-carboxylic acid dioxygenase (MHPCO), the seventh enzyme in degradation pathway I for pyridoxine, a free form of vitamin B(6). The gene was cloned and overexpressed in Escherichia coli cells co-transformed with chaperonin genes. The homogeneous recombinant enzyme showed similar enzymatic properties to the enzyme from Pseudomonas sp. MA-1. MHPCO was essential for the assimilation of pyridoxine in M. loti, but not for its growth in a nutrient-rich medium. From the infection experiment of a symbiotic plant Lotus japonicus with an M. loti mlr6788 gene disruptant, MHPCO was demonstrated to be dispensable for at least nodule formation on roots of seedlings in symbiosis.
Subject(s)
Escherichia coli/physiology , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Plant Roots/microbiology , Plant Roots/physiology , Proteobacteria/physiology , Cloning, Molecular/methods , Enzyme Activation , Enzyme Stability , Lotus/microbiology , Lotus/physiology , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/isolation & purification , Nitrogen Fixation/physiology , Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Symbiosis/physiologyABSTRACT
4-Pyridoxolactonase is involved in the degradation pathway for pyridoxine, a free form of vitamin B6. The gene (mlr6805) encoding the putative 4-pyridoxolactonase of nitrogen fixing symbiotic microorganism Mesorhizobium loti MAFF303099 has been identified based on the genome database. The gene was cloned and overexpressed in a cotransformant Escherichia coli cell. The recombinant enzyme was dimeric protein and contained one mole of Zn2+ per mole of subunit. The enzyme showed about 30% identity with various N-acylhomoserine lactone lactonases and metallo-beta-lactamases. The phylogram made with ClustalW shows that 4-pyridoxolactonase makes a cluster with Agrobacterium tumefaciens acyl-homoserine lactone lactonase. The alignment of amino acid sequences suggests that 4-pyridoxolactonase has three histidine residues probably involved in binding of Zn2+.
Subject(s)
Bacterial Proteins/genetics , Carboxylic Ester Hydrolases/genetics , Phylogeny , Rhizobiaceae/enzymology , Agrobacterium tumefaciens/enzymology , Agrobacterium tumefaciens/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Carboxylic Ester Hydrolases/metabolism , Cloning, Molecular , Molecular Sequence Data , Protein Subunits/genetics , Protein Subunits/metabolism , Rhizobiaceae/genetics , Sequence Analysis, Protein , Zinc/metabolismABSTRACT
The gene product of mll6785 of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 was identified as pyridoxine 4-oxidase, the first enzyme in the vitamin B6-degradation pathway. The gene was cloned and ligated into pET-21a+. Escherichia coli BL21(DE3) was co-transformed with the constructed plasmid plus pKY206 containing groESL genes encoding chaperonins. The overexpressed protein was purified to homogeneity by the ammonium sulfate fractionation and three chromatography steps. The enzymatic properties of the purified protein, such as K(m) values for pyridoxine (213+/-19 microM) and oxygen (78+/-10 microM), were compared to those of pyridoxine 4-oxidase from two bacteria with known vitamin B6-degradation pathway. M. loti grown in a Rhizobium medium showed the enzyme activity. The results suggest that M. loti also contains the degradation pathway of vitamin B6.
