Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5'-phosphate-dependent decarboxylase: a functional study.
The pyridoxal-5'-phosphate-dependent enzymes (B6 enzymes) are grouped into three main families named alpha, beta, and gamma. Proteins in the alpha and gamma families share the same fold and might be distantly related, while those in the beta family exhibit specific structural features. The rat aromatic L-amino acid decarboxylase (AADC; EC(4.1.1.28)) catalyzes the synthesis of two important neurotransmitters: dopamine and serotonin. It binds the cofactor pyridoxal-5'-phosphate and belongs to the alpha family. Despite the low level of sequence identity (approximately 10%) shared by the rat AADC and the sequences of the enzymes belonging to the B6 enzymes family, including the known three-dimensional structures, a multiple sequence alignment was deduced. A model was built using segments belonging to seven of the eleven known structures. By homology, and based on knowledge of the biochemistry of the aspartate aminotransferase, structurally and functionally important residues were identified in the rat AADC. Site-directed mutagenesis of the conserved residues D271, T246, and C311 was carried out in order to confirm our predictions and highlight their functional role. Mutation of D271A and D271N resulted in complete loss of enzyme activity, while the D271E mutant exhibited 2% of the wild-type activity. Substitution of T246A resulted in 5% of the wild-type activity while the C311A mutant conserved 42% of the wild-type activity. A functional model of the AADC is discussed in view of the structural model and the complementary mutagenesis and labelling studies.
Poupon
A
A
Laboratoire de Minéralogie-Cristallographie, CNRS, Université, Paris VI, France. apoupon@cea.fr
Jebai
F
F
Labesse
G
G
Gros
F
F
Thibault
J
J
Mornon
J P
JP
Krieger
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000737:chemistry* / Q000235:genetics; D006900:Hydroxylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D011732:Pyridoxal Phosphate; D001142:Aromatic-L-Amino-Acid Decarboxylases