Monoamine neurotransmitters as substrates for novel tick sulfotransferases, homology modeling, molecular docking, and enzyme kinetics.
Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17β-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (K(m) = 0.1-0.4 μM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine.
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States.
Emine Bihter
EB
Yalcin
Hubert
H
Stangl
Sivakamasundari
S
Pichu
Thomas N
TN
Mather
Roberta S
RS
King
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015306:Biogenic Monoamines / Q000737:chemistry / Q000378:metabolism*; D001681:Biological Assay; D019295:Computational Biology / Q000379:methods*; D004298:Dopamine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008193:Lyme Disease / Q000201:enzymology / Q000378:metabolism* / Q000635:transmission; D008969:Molecular Sequence Data; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000378:metabolism*; D009655:Octopamine / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013987:Ticks / Q000201:enzymology / Q000378:metabolism*
D015306:Biogenic Monoamines; D018377:Neurotransmitter Agents; D020033:Protein Isoforms; D009655:Octopamine; D015238:Sulfotransferases; D004298:Dopamine