Kinetic Characterization and Catalytic Mechanism of N-Acetylornithine Aminotransferase Encoded by slr1022 Gene from Synechocystis sp. PCC6803

Abstract

The enzyme encoded by slr1022 gene from Synechocystis sp. PCC6803 was reported to function as N-acetylornithine aminotransferase, γ-aminobutyric acid aminotransferase, and ornithine aminotransferase, which played important roles in multiple metabolic pathways. Among these functions, N-acetylornithine aminotransferase catalyzes the reversible conversion of N-acetylornithine to N-acetylglutamate-5-semialdehyde with PLP as cofactor, which is a key step in the arginine biosynthesis pathway. However, the investigation of the detailed kinetic characteristics and catalytic mechanism of Slr1022 has not been carried out yet. In this study, the exploration of kinetics of recombinant Slr1022 illustrated that Slr1022 mainly functioned as N-acetylornithine aminotransferase with low substrate specificity to γ-aminobutyric acid and ornithine. Kinetic assay of Slr1022 variants and the model structure of Slr1022 with N-acetylornithine-PLP complex revealed that Lys280 and Asp251 residues were the key amino acids of Slr1022. The respective mutation of the above two residues to Ala resulted in the activity depletion of Slr1022. Meanwhile, Glu223 residue was involved in substrate binding and it served as a switch between the two half reactions. Other residues such as Thr308, Gln254, Tyr39, Arg163, and Arg402 implicated a substrate recognition and catalytic process of the reaction. The results of this study further enriched the understanding of the catalytic kinetics and mechanism of N-acetylornithine aminotransferase, especially from cyanobacteria.

Biochemical and Structural Insights into a Thiamine Diphosphate-Dependent α-Ketoglutarate Decarboxylase from Cyanobacterium Microcystis aeruginosa NIES-843

“…The native molecular weights of MaKGD and MiKGD are determined to be 242.26 kDa and 243.70 kDa, respectively, by the gel filtration method, as reported previously [16,18]. Based on these results, it can be inferred that both MaKGD and MiKGD exist as tetramers in their native states since their molecular weights of subunits are 59.82 KDa.…”

“…The model structure of MaKGD was predicted by AlphaFold [37]. The validation of the MaKGD model structure was carried out using the method described in our previous report [18]. The protein-ligand/substrate docking was completed by AutoDockTools-1.5.7 [38].…”

Chlorophyll biosynthesis under the control of arginine metabolism

“… 28 Although it was proposed to function also as Orn 38 or gamma aminobutyric acid 39 aminotransferases, these activities are negligible compared to its AcOrn aminotransferase activity. 32 These results imply that ArgD-Gun4 participates in an Arg biosynthesis-dependent regulation of the tetrapyrrole pathway. ArgD bound Gun4 with a K d of 55.8 ± 11.5 nM ( Figure 1 C), which is close to the binding constant obtained by a fluorescence quenching measurement for the Gun4-ChlH complex (K d ∼10 nM).…”

“…The mass of f.ArgD (49.8 kDa) on the BN gel appeared at about 100 kDa, indicating a dimer consistent with other studies. 32 Given the well-established and critical role of Gun4 in Chl biosynthesis, 24 we focused on the interaction of Gun4 with enzymes involved in Arg metabolism (ArgD and CphB).…”

Arginine inhibits the arginine biosynthesis rate-limiting enzyme and leads to the accumulation of intracellular aspartate in Synechocystis sp. PCC 6803

“…The specific activity of the key enzyme in arginine biosynthesis, NAGK, is higher than 3 U/mg in the absence of arginine in Synechocystis 6803 (Bolay et al 2021 ). In addition, the activity of the arginine biosynthesis key enzyme of Synechocystis 6803, N -acetylornithine aminotransferase, for N -acetylornithine ( k cat / K m : 19.3 s − 1 mM − 1 ) was approximately 10-fold higher than that of Sy ArgG ( k cat / K m : 1.97 s − 1 mM − 1 ) (Li et al 2023 ; Table 1 ). Synechocystis 6803 cell crude extract experiments also show that N -acetylornithine aminotransferase activity is 2-fold higher than argininosuccinate synthetase activity (Liu and Yang 2014 ).…”