Identification of specific histidine residues and the carboxyl terminus are essential for serotonin N-acetyltransferase enzymatic activity.
Melatonin is synthesized in pinealocytes of the pineal gland and in photoreceptors of the retina. Synthesis rate from serotonin to melatonin is controlled by the rapid and dramatic enzymatic increase in darkness of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, EC 2.3.1.87) and hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The primary structure of these critical indoleamine enzymes is now known and the regulation of the enzyme catalysis can be examined. As a first step, the conserved cysteine (C) and histidine (H) residues were targeted for site-directed mutagenesis as potential amino acid residues involved in the N-acetylation reaction of AA-NAT. Our studies concluded that among 6 histidine (H) to alanine (A) mutations, three residues (H110A, H118A, H120A) within the AA-NAT protein showed little or no enzymatic activity, whereas the others (H28A, H70A, H125A) retained enzymatic activity, compared to the unaltered AA-NAT protein. Cysteine to alanine mutations, C37A and C177A, had no significant effect on the AA-NAT enzymatic activity; however, C61A had a four-fold increase in K(m) for acetyl CoA and an altered sensitivity to the thiol modification chemical, N-ethylmaleimide (NEM), implying that C61 may participate in the acetyl CoA binding. Further studies examined the AA-NAT enzyme regulation of the highly conserved carboxyl terminus. When 12 terminal amino acid residues were deleted systematically from the carboxyl terminus of the 205 amino acid residue AA-NAT protein, enzyme activity was retained. However, further residue deletion resulted in enzyme activity plummeting, implicating that the essential information either for the correct structural folding into an active enzyme form or for enzyme stability is in the 193 residues. To test the relative importance of the AA-NAT carboxyl terminal region, a single leucine (L) was altered to alanine (A) or proline (P). Both mutants, either L193A or L193P, had a marked decrease in AA-NAT enzymatic activity and a decrease in thermal stability, suggesting the leucine, in addition to the cysteine and histidine residues, is involved in either enzyme catalysis or stability. In light of the recently reported three-dimensional structure of AA-NAT (17,18), the site-directed mutagenesis data demonstrate experimentally the importance of essential amino acid residues for acetyl CoA binding and AA-NAT activation.
Craft
C M
CM
The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, Department of Cell and Neurobiology, Keck School of Medicine of the University of Southern California School, 1333 San Pablo Street, BMT 401, Los Angeles, CA, USA. ccraft@hsc.usc.edu
Zhan-Poe
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D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D004795:Enzyme Stability; D017353:Gene Deletion; D006639:Histidine / Q000032:analysis* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017386:Sequence Homology, Amino Acid
D006639:Histidine; D000105:Acetyl Coenzyme A; D001191:Arylamine N-Acetyltransferase; D003545:Cysteine