Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions |
| |
| Affiliation: | 1. Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA24060, United States;2. Laboratory of Tropical Veterinary Medicine and Vector Biology, Hainan Key Laboratory of Sustainable Utilization of Tropical Bioresources, Institute of Agriculture and Forestry, Hainan University, Haikou, 570228, Hainan, China;1. ZBSA, Center for Biological Systems Analysis, University of Freiburg, 79104 Freiburg, Germany;2. Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;1. National Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS, 38677, USA;2. Department of Applied Chemistry and Pharmacy, School of Chemical Engineering and Environment, Beijing Institute of Technology, Beijing, 100081, PR China;3. Division of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, MS, 38677, USA;4. TCM and Ethnomedicine Innovation, Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, PR China;5. Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria, 0001, South Africa;1. Department of Medical Biochemistry, Medical University of Lodz, ul. Mazowiecka 6/8, 92-215 Lodz, Poland;2. Department of General Biophysics, University of Lodz, ul. Pomorska 141/143, 90-236 Lodz, Poland;3. c-LEcta GmbH, Perlickstraße 5, 04-103 Leipzig, Germany;4. Laboratory of Theory of Biopolymers, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland;1. Department of Food and Life Sciences, Ibaraki University, 3-21-1 Chuo, Ami-machi, Inashiki, Ibaraki 300-0393, Japan;2. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-city, Chiba 277-8567, Japan |
| |
| Abstract: | In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO2, NH3, and H2O2. This contrasts to the typical DDC-catalyzed reaction, which produces CO2 and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H2O2 in the process. Biochemical assessment established that H2O2, formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H2O; thereby avoiding oxidative stress by H2O2. Results of our structural and functional analyses provide a reasonable explanation of mechanisms involved in DHPA synthase-mediated reactions. Based on the key active site residue Asn192, identified in Drosophila DHPA synthase, we were able to distinguish all available insect DHPA synthases from DDC sequences primarily. |
| |
| Keywords: | Decarboxylase Aromatic acetaldehyde synthase Decarboxylation Oxidative deamination |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
