Amino acid racemase responsible for the biosynthesis of D-aspartate and D-serine signaling molecules in Aplysia californica central nervous system

摘要

ABSTRACTWhile dogma states that animals use L-amino acids, several D-amino acids have been found in animals including humans since the 1980s. Two D-amino acids have attracted significant research interests because of their cell-cell signaling functions in animal nervous systems. D-serine (D-Ser) can affect the activity and plasticity of N-methyl-D-aspartate (NMDA) receptor, a molecule that forms the basis of learning and memory in the brain. D-aspartate (D-Asp) is found in neuroendocrine and endocrine tissues, potentially playing roles in hormone synthesis and/or release. However, many details on the physiological functions of D-Ser and D-Asp and their signaling pathways remain unknown. Extensive studies are needed to fully understand the roles of these two putative neuronal signaling molecules and their effects on the animal brain and endocrine functions. D-Ser and D-Asp biosynthetic enzymes are essential to study the physiological functions of the D-amino acids in animal nervous systems. However, the knowledge of such enzymes has been limited, especially the characterization of D-Asp synthetic enzymes. Although the first enzyme that can make D-Ser from L-Ser was isolated from animal brain tissue in 1999, the first nervous system associated D-Asp synthetic enzyme was not described until 2010. The lack of the knowledge of a D-Asp biosynthetic enzyme has significantly hindered the research of D-Asp physiological function in animal nervous system. The molecular mechanism underlying D-Asp action is still not clear. In invertebrates, high levels of D-Asp had been found in central nervous systems (CNS) of some marine mollusks including Aplysia californica, a model animal for cellular and systemic neuroscience study, and D-Asp showed neurotransmitter behaviors in Aplysia CNS, indicating D-Asp signaling pathway is conserved among animal kingdoms. Thus, studying D-Asp in Aplysia can provide valuable insights into mammalian system function.As the biosynthetic pathway of D-Asp in invertebrate is not known, one significant goal of this project has been to uncover the biosynthetic enzyme responsible for the biosynthesis of D-Asp in cerebral ganglion in Aplysia CNS. Using combining bioinformatics, molecular biology and biochemical methods, a d-amino acid racemase 1 (DAR1) has been discovered, cloned and characterized from the Aplysia CNS. The biosynthesis of D-Ser and D-Asp in Aplysia CNS cerebral ganglion F/C neuronal clusters via this enzyme has been characterized; these are the regions where high levels of D-Asp were previously detected. DAR1 is the first serine racemase and the first nervous system associated aspartate racemase described from an invertebrate system, and it is also the first enzyme with a dual serine and aspartate activity described in a eukaryotic system. The enzyme thus becomes a valuable model to study D-Ser and D-Asp physiological functions in a well-characterized model system, to investigate D-Ser and D-Asp signaling pathways and their interactions, and to examine the structure-function relationships of pyridoxal-5???-phosphate (PLP) dependent amino acid racemase.