Tài liệu Báo cáo khoa học: Tissue expression and biochemical characterization of human 2-amino

Tissue expression and biochemical characterization

of human 2-amino 3-carboxymuconate 6-semialdehyde

decarboxylase, a key enzyme in tryptophan catabolism

Lisa Pucci*, Silvia Perozzi*, Flavio Cimadamore, Giuseppe Orsomando and Nadia Raffaelli

Istituto di Biotecnologie Biochimiche, Universita` Politecnica delle Marche, Ancona, Italy

In mammals, tryptophan exceeding basal requirement

for protein and serotonin synthesis, is oxidized via

indole-ring cleavage through the kynurenine pathway,

consisting of several enzymatic reactions leading to

2-amino 3-carboxymuconate 6-semialdehyde (ACMS)

(Fig. 1) [1,2]. ACMS can be decarboxylated to

2-aminomuconate 6-semialdehyde (AMS) by the

enzyme ACMS decarboxylase (ACMSD, EC 4.1.1.45),

or it can undergo spontaneous pyridine ring closure to

form quinolinate, an essential precursor for de novo

NAD synthesis. AMS can be routed to the citric

acid cycle via the glutarate pathway, or converted

nonenzymatically to picolinate. By catalyzing ACMS

decarboxylation, ACMSD thus diverts ACMS from

NAD synthesis, channeling tryptophan towards

complete oxidation or conversion to picolinate.

By determining picolinate and quinolinate forma￾tion, ACMSD directly participates in the cellular pro￾cesses regulated by these molecules. Quinolinate is a

neurotoxic tryptophan metabolite, whose action has

been ascribed to N-methyl-D-aspartate receptors

activation and to its ability to generate free radicals

Keywords

ACMSD; NAD biosynthesis; picolinate;

quinolinate; tryptophan catabolism

Correspondence

N. Raffaelli, Istituto di Biotecnologie

Biochimiche, Universita` Politecnica delle

Marche, Via Ranieri, 60131 Ancona, Italy

Fax: +39 712204677

Tel: +39 712204682

E-mail: [email protected]

*These authors contributed equally to this

paper

(Received 16 November 2006, accepted

6 December 2006)

doi:10.1111/j.1742-4658.2007.05635.x

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 quinoli￾nate and picolinate formation. ACMSD is therefore an attractive therapeu￾tic 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 Km of 6.5 lm, and a kcat of 1.0 s)1

.

ACMSD I is inhibited by quinolinic acid, picolinic acid and kynurenic

acid, and it is activated slightly by Fe2+ and Co2+. Site-directed mutagen￾esis 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.

Abbreviations

ACMS, 2-amino 3-carboxymuconate 6-semialdehyde; ACMSD, ACMS decarboxylase; AMS, 2-aminomuconate 6-semialdehyde.

FEBS Journal 274 (2007) 827–840 ª 2007 The Authors Journal compilation ª 2007 FEBS 827