Tissue expression and biochemical characterization of human 2-amino 3-carboxymuconate 6-semialdehyde decarboxylase, a key enzyme in tryptophan catabolism.
2-amino 3-carboxymuconate 6-semialdehyde decarboxylase (ACMSD, EC 4.1.1.45) plays a key role in tryptophan catabolism. By diverting 2-amino 3-carboxymuconate semialdehyde from quinolinate production, the enzyme regulates NAD biosynthesis from the amino acid, directly affecting quinolinate and picolinate formation. ACMSD is therefore an attractive therapeutic target for treating disorders associated with increased levels of tryptophan metabolites. Through an isoform-specific real-time PCR assay, the constitutive expression of two alternatively spliced ACMSD transcripts (ACMSD I and II) has been examined in human brain, liver and kidney. Both transcripts are present in kidney and liver, with highest expression occurring in kidney. In brain, no ACMSD II expression is detected, and ACMSD I is present at very low levels. Cloning of the two cDNAs in yeast expression vectors and production of the recombinant proteins, revealed that only ACMSD I is endowed with enzymatic activity. After purification to homogeneity, this enzyme was found to be a monomer, with a broad pH optimum ranging from 6.5 to 8.0, a K(m) of 6.5 microM, and a k(cat) of 1.0 s(-1). ACMSD I is inhibited by quinolinic acid, picolinic acid and kynurenic acid, and it is activated slightly by Fe(2+) and Co(2+). Site-directed mutagenesis experiments confirmed the catalytic role of residues, conserved in all ACMSDs so far characterized, which in the bacterial enzyme participate directly in the metallocofactor binding. Even so, the properties of the human enzyme differ significantly from those reported for the bacterial counterpart, suggesting that the metallocofactor is buried deep within the protein and not as accessible as it is in bacterial ACMSD.
Pucci
Lisa
L
Istituto di Biotecnologie Biochimiche, Università Politecnica delle Marche, Ancona, Italy.
Perozzi
Silvia
S
Cimadamore
Flavio
F
Orsomando
Giuseppe
G
Raffaelli
Nadia
N
D000595:Amino Acid Sequence; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D017378:Quinolinic Acid / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D014364:Tryptophan; D002262:Carboxy-Lyases; C024396:aminocarboxymuconate-semialdehyde decarboxylase; D017378:Quinolinic Acid