Backbone resonance assignments of dopamine N-acetyltransferase in free and cofactor-bound states

Abstract

Dopamine N-acetyltransferase (Dat), belonging to the GCN5-related N-acetyltransferase (GNAT) superfamily, is an arylalkylamine N-acetyltransferase (AANAT) that is involved in insects neurotransmitter inactivation and the development of insect cuticle sclerotization. By using the cofactor acetyl coenzyme A (Ac-CoA) as an acetyl group donor, Dat produces acetyl-dopamine through the reaction with dopamine. Although AANATs share similar structural features with the GNAT family, they have low sequence identities among insect AANATs (~ 40%) and between insect AANATs and vertebrate AANATs (~ 12%). A common noticed feature in GNATs is the Ac-CoA-binding induced conformational change, and this is important for further selection and catalysis of its substrate. In AANATs, the conformational changes help the sequential binding mechanism. Here, we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments of the 24 kDa Dat from Drosophila melanogaster in the free and Ac-CoA-bound states, and the chemical shift differences revealed a significant conformational change in the α1 region of Dat. These assignments provide a foundation for further investigations of the catalysis and structural regulation of Dat in solution.

Keywords: AANAT; Arylalkylamine; Dopamine N-acetyltransferase (Dat); GNAT; N-acetyltransferase; NMR.

Fig. 1

Assignments of the 2D ¹⁵N–¹H HSQC spectra of apo-Dat and the Dat/Ac-CoA complex recorded at 850 MHz and 293 and 298 K, respectively. Horizontal lines highlight signals from side chains, and black boxes highlight the residues for which two conformations are observed

Fig. 2

Chemical shift predicted order parameters (S²; upper panel) and secondary structures (bottom panel) using TALOS-N index based on the backbone N, H, Cα, C’ and side chain Cβ chemical shift values. Predicted helices are shown by red bars, and predicted strands by teal bars

Fig. 3

Chemical shift differences, Δδ, between the apo-Dat and Ac-CoA-bound states show significant changes in the α1, β4 and α5-7 regions (left panel). The red line indicates the mean value of all the chemical shift difference values. The black line and dashed line denote cut-offs for significance at 1 standard deviation (1σ) and 2 standard deviations (2σ), respectively. Red circles represent the binding residues for Ac-CoA, which are L146, V148, R153, G154, G156, A158, V179, V189, and K192 as analyzed by LigPlot + (Laskowski & Swindells 2011). Yellow bars represent proline residues that do not have NH peaks in 2D ¹⁵N–¹H HSQC spectra. Blue bars represent unassigned residues in apo-Dat or Dat/Ac-CoA, which may be caused by peak broadening. Chemical shift differences of residues with significant changes were mapped onto the structure of Dat (PDB code: 3TE4) (right panel)

Fig. 4

Overlay of HSQC Spectra of apo-Dat at 20 °C and 25 °C

Fig. 5

Ile, Leu, and Val selective unlabeling of apo-Dat and Dat/Ac-CoA. Missing amide signals in the ¹H-¹⁵N-HSQC which resulted from specific unlabeling are indicated