Background: Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.
Methods: HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.
Results: HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37°C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5'-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.
Conclusions: These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.
General significance: The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.
Keywords: C7orf52; Ectotherm; HISAT; Histidine N-acetyltransferase; MALDI-TOF-MS; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry; N-acetyltransferase 16; NAH; NAT16; Nα-acetylhistidine; RACE; RT-PCR; histidine N-acetyltransferase; rapid amplification of cDNA ends; reverse transcription-PCR.
© 2013.