ABSTRACT
Succinic semialdehyde dehydrogenases (SSADH) of cyanobacteria played a pivotal role in completing the cyanobacterial tricarboxylic acid cycle. The structural information of cofactor preference and catalysis for SSADH from cyanobacteria is currently available. However, the detailed kinetics of SSADH from cyanobacteria were not characterized yet. In this study, an all3556 gene encoding SSADH from Anabaena sp. PCC7120 (ApSSADH) was amplified and the recombinant ApSSADH was purified homogenously. Kinetic analysis showed that ApSSADH was an NADP+-dependent SSADH, which utilized NADP+ and succinic semialdehyde (SSA) as its preferred substrates and the activity of ApSSADH was inhibited by its substrate of SSA. At the same time, the Ser157 residue was found to function as the determinant of cofactor preference. Further study demonstrated that activity and substrate inhibition of ApSSADH would be greatly reduced by the mutation of the residues at the active site. Bioinformatic analysis indicated that those residues were highly conserved throughout the SSADHs. To our knowledge this is the first report exploring the detailed kinetics of SSADH from cyanobacteria.
Subject(s)
Anabaena/metabolism , NADP/chemistry , NADP/metabolism , Succinate-Semialdehyde Dehydrogenase/chemistry , Succinate-Semialdehyde Dehydrogenase/metabolism , Anabaena/genetics , Binding Sites , Catalysis , Catalytic Domain , Coenzymes , Enzyme Activation , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Metabolic Networks and Pathways , Models, Molecular , Molecular Conformation , Molecular Weight , Mutation , Protein Binding , Recombinant Proteins , Substrate Specificity , Succinate-Semialdehyde Dehydrogenase/genetics , Succinate-Semialdehyde Dehydrogenase/isolation & purificationABSTRACT
The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min(-1) mg(-1), using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.
Subject(s)
Aspergillus niger/enzymology , Fungal Proteins/metabolism , Succinate-Semialdehyde Dehydrogenase/metabolism , gamma-Aminobutyric Acid/analogs & derivatives , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Aspergillus niger/metabolism , Benzaldehydes/metabolism , Benzaldehydes/pharmacology , Binding, Competitive , Biocatalysis/drug effects , Fungal Proteins/isolation & purification , Kinetics , Mycelium/enzymology , Mycelium/metabolism , NAD/metabolism , NAD/pharmacology , Protein Binding , Substrate Specificity , Succinate-Semialdehyde Dehydrogenase/isolation & purification , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacologyABSTRACT
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 function of the mlr6787 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 2-(acetamidomethylene)succinate hydrolase, an enzyme involved in the catabolism of pyridoxal 5'-phosphate (vitamin B 6). This enzyme was overexpressed in Escherichia coli, purified to homogeneity, and characterized. 2-(Acetamidomethylene)succinate hydrolase catalyzes the hydrolysis of 2-(acetamidomethylene)succinate to yield succinic semialdehyde, acetic acid, carbon dioxide, and ammonia. The k cat and K M for this reaction were 0.6 s (-1) and 143 microM, respectively. The enzyme was shown to utilize the E isomer of 2-(acetamidomethylene)succinate.
