Crystal structure and substrate specificity of Drosophila 3,4-dihydroxyphenylalanine decarboxylase

Abstract

Background: 3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses.

Principal findings: In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine.

Conclusions: The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

Figure 1. Effect of temperature and pH on drDDC activity.

The activities of recombinant drDDC at different temperatures (A) and at different pH values (B).

Figure 2. Overall structure and schematic view of one subunit of drDDC.

A, A schematic representation of the structure of drDDC dimer. B, The schematic view of a monomer. The cofactor (LLP) is included in stick. Three parts, large domain (green), small domain (blue) and N-terminal part (pink) are labeled.

Figure 3. The drDDC active site.

A stereo view of the active site in the drDDC structure. The LLP and the protein residues within a 4 Å distance of the cofactor are shown. Only the 2F _o - F _c electron density map covering the LLP is shown contoured at the 1.8 sigma. Hydrogen bonds are shown in dashed lines.

Figure 4. Superposition of drDDC structure onto pig DDC structure.

The protein portions within 12 Å of the active center are shown in the schematic representation in stereo. Pig DDC and drDDC chain As are colored in cyan and magenta, respectively; and their chain Bs are colored in deep teal and brown, respectively.

Figure 5. Molecular structures of tested chemicals and decarboxylation activity of drDDC WT and three mutants towards different aromatic amino acid substrates.

A, Molecular structures of tyr (tyrosine), o-tyr, m-tyr, DOPA, trp (tryptophan) and 5-HTP; B, Decarboxylation activity. A typical reaction mixture of 100 µl consists of 5 µg of purified drDDC WT or any mutants, 1 mM of DOPA, 5-HTP, m-tyr, o-tyr, trp, tyr or d-DOPA, and 40 mM PLP in 150 mM potassium phosphate buffer, pH 7.0. Vertical axis shows the specific activity of the enzymes to each substrate (trp, tyr, and d-DOPA are not shown because none of the proteins showed any activity to them), and horizontal axis shows the drDDC WT and mutant protein names.

Figure 6. The roles of T82 and H192.

A, Superposition of drDDC structure onto pig DDC structure shows that T82 of drDDC is a putative substrate binding residue. Residues from pig DDC are colored in cyan and those from drDDC are colored in magenta. B, Alignment of drDDC, pig DDC, Drosophila tyrosine decarboxylase-1 (drTDC-1) and Drosophila tyrosine decarboxylase-2 (drTDC-2). The corresponding residues of T82 and H192 are labeled. C, Superposition of drDDC structure onto pig DDC structure shows the H192 of drDDC is a putative substrate and cofactor binding residue. Residues from pig DDC are colored in cyan and those from drDDC are colored in magenta.