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Drosophila tan encodes a novel hydrolase required in pigmentation and vision.

True JR, Yeh SD, Hovemann BT, Kemme T, Meinertzhagen IA, Edwards TN, Liou SR, Han Q, Li J - PLoS Genet. (2005)

Bottom Line: We characterized two tan-like P-element insertions that failed to complement classical tan mutations.Both P insertions showed abnormally low transcription of the CG12120 mRNA.We conclude that D. melanogaster CG12120 corresponds to tan.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolution, State University of New York, Stony Brook, New York, United States of America. jrtrue@life.bio.sunysb.edu

ABSTRACT
Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-beta-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5' untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function.

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The CG12120 Gene Product Possesses NBAD Hydrolase and N-β-Alanyl Histamine (Carcinine) Hydrolase ActivitiesHPLC-ED chromatograms from activity assays.(A) Lysate from control (recombinant AGT) transfected Sf9 cells exhibited no activity. Oxidation product (OP) peak denotes uncharacterized presumptive oxidation product of NBAD present in NBAD substrate in all experiments.(B) NBAD incubated with CG12120 protein purified from Sf9 cells expressing CG12120 baculovirus construct after 20 min incubation with NBAD. Dopamine (DA) peak is evident.(C) Same experiment as in (B) but after 50 min incubation, showing increased accumulation of dopamine and depletion of NBAD.(D) Control HPLC chromatogram containing dopamine and NBAD standards.(E) Extract from control (recombinant AGT) transfected Sf9 cells exhibits no production of β-alanine and histamine when incubated with carcinine (CA). Unknown contaminant (UC) peak denotes an unknown contaminant in carcinine. β-mercaptoethanol (BME) peak denotes β-mercaptoethanol present in the reaction solution.(F) Extract from Sf9 cells transfected with CG12120 baculovirus incubated with carcinine demonstrates production of histamine (HA) and β-alanine (BA). Under the applied conditions, essentially all carcinine was hydrolyzed to histamine and β-alanine.(G) Control HPLC chromatogram containing β-mercaptoethanol, carcinine, histamine, and β-alanine standards. β-alanine and histamine detection were enabled by OPT conjugation (see Materials and Methods).
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pgen-0010063-g004: The CG12120 Gene Product Possesses NBAD Hydrolase and N-β-Alanyl Histamine (Carcinine) Hydrolase ActivitiesHPLC-ED chromatograms from activity assays.(A) Lysate from control (recombinant AGT) transfected Sf9 cells exhibited no activity. Oxidation product (OP) peak denotes uncharacterized presumptive oxidation product of NBAD present in NBAD substrate in all experiments.(B) NBAD incubated with CG12120 protein purified from Sf9 cells expressing CG12120 baculovirus construct after 20 min incubation with NBAD. Dopamine (DA) peak is evident.(C) Same experiment as in (B) but after 50 min incubation, showing increased accumulation of dopamine and depletion of NBAD.(D) Control HPLC chromatogram containing dopamine and NBAD standards.(E) Extract from control (recombinant AGT) transfected Sf9 cells exhibits no production of β-alanine and histamine when incubated with carcinine (CA). Unknown contaminant (UC) peak denotes an unknown contaminant in carcinine. β-mercaptoethanol (BME) peak denotes β-mercaptoethanol present in the reaction solution.(F) Extract from Sf9 cells transfected with CG12120 baculovirus incubated with carcinine demonstrates production of histamine (HA) and β-alanine (BA). Under the applied conditions, essentially all carcinine was hydrolyzed to histamine and β-alanine.(G) Control HPLC chromatogram containing β-mercaptoethanol, carcinine, histamine, and β-alanine standards. β-alanine and histamine detection were enabled by OPT conjugation (see Materials and Methods).

Mentions: The tan gene product is predicted to encode a multifunctional hydrolase that catalyzes the hydrolysis of NBAD into β-alanine and dopamine, and the hydrolysis of carcinine (N-β-alanyl histamine) into β-alanine and histamine, respectively. To test whether CG12120 possesses these predicted activities, we produced recombinant CG12120 protein using a baculovirus expression system in insect cell culture (Figure 4). After soluble proteins from either uninfected Spodoptera frugiperda (Sf9) insect cells or Sf9 cells infected with an alanine glyoxylate transaminase (AGT) recombinant baculovirus were mixed into a NBAD solution. Production of dopamine in the reaction mixture was not observed (Figure 4A), suggesting that Sf9 cells do not have a protein capable of mediating NBAD hydrolysis, and infection of baculovirus itself also did not stimulate the production of a protein with NBAD-hydrolyzing activity (data not shown). In contrast, when soluble proteins from CG12120 recombinant baculovirus-infected cells were mixed into a NBAD solution, accumulation of dopamine in the reaction mixture was observed and the amounts of dopamine produced in the reaction mixture were approximately proportional to the applied incubation periods (Figure 4B and 4C). During hydrolysis, an equal amount of β-alanine was produced in the reaction mixture (data not shown), but could not be detected electrochemically, because β-alanine is not electrochemically active.


Drosophila tan encodes a novel hydrolase required in pigmentation and vision.

True JR, Yeh SD, Hovemann BT, Kemme T, Meinertzhagen IA, Edwards TN, Liou SR, Han Q, Li J - PLoS Genet. (2005)

The CG12120 Gene Product Possesses NBAD Hydrolase and N-β-Alanyl Histamine (Carcinine) Hydrolase ActivitiesHPLC-ED chromatograms from activity assays.(A) Lysate from control (recombinant AGT) transfected Sf9 cells exhibited no activity. Oxidation product (OP) peak denotes uncharacterized presumptive oxidation product of NBAD present in NBAD substrate in all experiments.(B) NBAD incubated with CG12120 protein purified from Sf9 cells expressing CG12120 baculovirus construct after 20 min incubation with NBAD. Dopamine (DA) peak is evident.(C) Same experiment as in (B) but after 50 min incubation, showing increased accumulation of dopamine and depletion of NBAD.(D) Control HPLC chromatogram containing dopamine and NBAD standards.(E) Extract from control (recombinant AGT) transfected Sf9 cells exhibits no production of β-alanine and histamine when incubated with carcinine (CA). Unknown contaminant (UC) peak denotes an unknown contaminant in carcinine. β-mercaptoethanol (BME) peak denotes β-mercaptoethanol present in the reaction solution.(F) Extract from Sf9 cells transfected with CG12120 baculovirus incubated with carcinine demonstrates production of histamine (HA) and β-alanine (BA). Under the applied conditions, essentially all carcinine was hydrolyzed to histamine and β-alanine.(G) Control HPLC chromatogram containing β-mercaptoethanol, carcinine, histamine, and β-alanine standards. β-alanine and histamine detection were enabled by OPT conjugation (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1285064&req=5

pgen-0010063-g004: The CG12120 Gene Product Possesses NBAD Hydrolase and N-β-Alanyl Histamine (Carcinine) Hydrolase ActivitiesHPLC-ED chromatograms from activity assays.(A) Lysate from control (recombinant AGT) transfected Sf9 cells exhibited no activity. Oxidation product (OP) peak denotes uncharacterized presumptive oxidation product of NBAD present in NBAD substrate in all experiments.(B) NBAD incubated with CG12120 protein purified from Sf9 cells expressing CG12120 baculovirus construct after 20 min incubation with NBAD. Dopamine (DA) peak is evident.(C) Same experiment as in (B) but after 50 min incubation, showing increased accumulation of dopamine and depletion of NBAD.(D) Control HPLC chromatogram containing dopamine and NBAD standards.(E) Extract from control (recombinant AGT) transfected Sf9 cells exhibits no production of β-alanine and histamine when incubated with carcinine (CA). Unknown contaminant (UC) peak denotes an unknown contaminant in carcinine. β-mercaptoethanol (BME) peak denotes β-mercaptoethanol present in the reaction solution.(F) Extract from Sf9 cells transfected with CG12120 baculovirus incubated with carcinine demonstrates production of histamine (HA) and β-alanine (BA). Under the applied conditions, essentially all carcinine was hydrolyzed to histamine and β-alanine.(G) Control HPLC chromatogram containing β-mercaptoethanol, carcinine, histamine, and β-alanine standards. β-alanine and histamine detection were enabled by OPT conjugation (see Materials and Methods).
Mentions: The tan gene product is predicted to encode a multifunctional hydrolase that catalyzes the hydrolysis of NBAD into β-alanine and dopamine, and the hydrolysis of carcinine (N-β-alanyl histamine) into β-alanine and histamine, respectively. To test whether CG12120 possesses these predicted activities, we produced recombinant CG12120 protein using a baculovirus expression system in insect cell culture (Figure 4). After soluble proteins from either uninfected Spodoptera frugiperda (Sf9) insect cells or Sf9 cells infected with an alanine glyoxylate transaminase (AGT) recombinant baculovirus were mixed into a NBAD solution. Production of dopamine in the reaction mixture was not observed (Figure 4A), suggesting that Sf9 cells do not have a protein capable of mediating NBAD hydrolysis, and infection of baculovirus itself also did not stimulate the production of a protein with NBAD-hydrolyzing activity (data not shown). In contrast, when soluble proteins from CG12120 recombinant baculovirus-infected cells were mixed into a NBAD solution, accumulation of dopamine in the reaction mixture was observed and the amounts of dopamine produced in the reaction mixture were approximately proportional to the applied incubation periods (Figure 4B and 4C). During hydrolysis, an equal amount of β-alanine was produced in the reaction mixture (data not shown), but could not be detected electrochemically, because β-alanine is not electrochemically active.

Bottom Line: We characterized two tan-like P-element insertions that failed to complement classical tan mutations.Both P insertions showed abnormally low transcription of the CG12120 mRNA.We conclude that D. melanogaster CG12120 corresponds to tan.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolution, State University of New York, Stony Brook, New York, United States of America. jrtrue@life.bio.sunysb.edu

ABSTRACT
Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-beta-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5' untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function.

Show MeSH
Related in: MedlinePlus