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Previous studies have shown that several d-amino acids are widely present in plants, and serine racemase (SerR), which synthesizes d-serine in vivo, has already been identified from three plant species. However, the full picture of the d-amino acid synthesis pathway in plants is not well understood. To clarify the distribution of amino acid racemases in plants, we have cloned, expressed and characterized eight SerR homologous genes from five plant species, including green alga. These SerR homologs exhibited racemase activity towards serine or aspartate and were identified on the basis of their maximum activity as SerR or aspartate racemase (AspR). The plant AspR gene is identified for the first time from Medicago truncatula, Manihot esculenta, Solanum lycopersicum, Sphagnum girgensohnii and Spirogyra pratensis. In addition to the AspR gene, three SerR genes are identified in the former three species. Phylogenetic tree analysis showed that SerR and AspR are widely distributed in plants and form a serine/aspartate racemase family cluster. The catalytic efficiency (k cat /K m ) of plant AspRs was more than 100 times higher than that of plant SerRs, suggesting that d-aspartate, as well as d-serine, can be synthesized in vivo by AspR. The amino acid sequence alignment and comparison of the chromosomal gene arrangement have revealed that plant AspR genes independently evolved from SerR in each ancestral lineage of plant species by gene duplication and acquisition of two serine residues at position 150 to 152., (Copyright © 2019 Elsevier Ltd. All rights reserved.)