1887

Abstract

Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N-fixing cyanobacteria. In this report, five putative AspAT genes (, , , and ) were identified in the N-fixing heterocystous cyanobacterium sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H) were overexpressed in and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed . Alr4853-H showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and was further identified to be a Fox gene (requiring fixed N for growth in the presence of oxygen). Four out of five P transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that expression was increased by 2.3-fold after 24 h of N deprivation. Taken together, these findings add to our understanding of the role of AspATs in N-fixing within heterocystous cyanobacteria.

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2015-06-01
2020-01-23
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