Abstract

N-acetylornithine aminotransferase (AcOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a key transamination step in arginine biosynthesis. In cyanobacteria, arginine metabolism is closely associated with nitrogen assimilation and storage, yet biochemical information on cyanobacterial AcOATs remains limited. In this study, the AcOAT encoded by the cce_3094 gene from Crocosphaera subtropica ATCC 51142 (CsAcOAT) was cloned, heterologously expressed, purified, and systematically characterized. Recombinant CsAcOAT was obtained as a soluble protein with an apparent molecular mass of approximately 43 kDa. Steady-state kinetic analysis showed that CsAcOAT catalyzed transamination between N-acetylornithine (AcOrn) and α-ketoglutarate (α-KG), with apparent KM values of 0.17 ± 0.03 mM for AcOrn and 0.020 ± 0.003 mM for α-KG, indicating a higher affinity for α-KG. The enzyme exhibited optimal activity at pH 8.5 and 30 °C, retained relatively high activity over a broad temperature range of 0–50 °C, and was activated by Zn2+ and Co2+ but inhibited by Ni2+. Structural analysis based on homology modeling, molecular docking, and molecular dynamics simulations suggested a conserved PLP-dependent aminotransferase fold and a stable binding mode for the PLP-AcOrn complex in the active-site pocket. Site-directed mutagenesis further demonstrated that Gly114, Asp239, Lys268, and Thr296 are indispensable for catalytic activity, whereas Ser113, Ala115, and Gln242 make important contributions to catalytic turnover and cofactor-assisted catalysis. These results provide biochemical and structural characterization of CsAcOAT, expand current knowledge of cyanobacterial AcOATs, and offer a useful basis for future studies on arginine metabolism and nitrogen storage in diazotrophic cyanobacteria.

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