Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase

Butrin, Arseniy; Butrin, Anastassiya; Wawrzak, Z.; Moran, Graham R.; Liu, Dali

doi:10.1016/j.jbc.2022.101969

Cited by 3 publications

(3 citation statements)

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“…However, the K M of α-KG with the enzyme and the catalytic rates of E223A and E223S were close to those of the wild-type protein ( Table 2 ). These results proved that Glu223 residue was crucial for the binding of substrate AcOrn with enzyme and less important for the binding of substrate α-KG with enzyme, which was observed for the OAT from humans [ 21 ]. Similarly, the K M of AcOrn with enzyme would increase significantly by 4100 and 2080 folds for R163A and R402A mutants, respectively, compared to that of the wild-type protein.…”

Section: Resultsmentioning

confidence: 77%

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

Bai

Wang

et al. 2023

IJMS

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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.

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Section: Resultsmentioning

confidence: 77%

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

Bai

Wang

et al. 2023

IJMS

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“…Interestingly, a previously published crystal structure of 5-aminovaleric acid complexed with h OAT displayed a nearly identical stabilization of the carboxylate with Tyr55, Arg180, and a water molecule (Figure S6). 5-Aminovaleric acid is a slow-binding alternate substrate for h OAT and has been reported to bind in a different orientation compared with ornithine due to lack of an α-amino group. Tyr55 plays a distinct role in the accommodation of the charged α-amino group of ornithine in the active site of h OAT when compared to the hydrophobic residue Phe351 in GABA-AT .…”

Section: Resultsmentioning

confidence: 99%

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase

Devitt,

Vargas,

Zhu

et al. 2024

ACS Chem. Biol.

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Human ornithine aminotransferase (hOAT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19 F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

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“…They found that analogue 10b was a high selectivity and potency inhibitor for OAT. By using stopped‐flow spectrophotometry and X‐ray crystallography, Butrin et al 16 demonstrated the pH dependency of the human OAT multistep reaction mechanism and elucidated the roles of ornithine α‐amino and δ‐amino groups in substrate recognition and in facilitating catalytic turnover, which provide clues for the design of mechanism‐based inactivators against human OAT. Our GSEA results revealed that two amino acid metabolic pathways, aspartate aminotransferase and alanine aminotransferase, were activated in CPS1 high expression gastric cancer patients, which suggested that OAT was a promising target in treatment of gastric cancer.…”

Section: Discussionmentioning

confidence: 99%

Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer

Jiang

Chen

Luo

et al. 2022

Clinical Laboratory Analysis

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Objective The sensitivity and specificity of current biomarkers for gastric cancer were insufficient. The aim of the present study was to screen novel biomarkers and determine the diagnostic values of ornithine aminotransferase (OAT) and carbamoyl phosphate synthetase 1 (CPS1) for detecting gastric cancer. Methods With stable isotope tags, we labelled an initial discovery group of four paired gastric cancer tissue samples and identified with LC‐ESI‐MS/MS. A validation group of 159 gastric cancer samples and 30 healthy controls were used to validate the candidate targets. GSEA was used to explore the pathways activated in gastric cancer. Results Four hundred and thirty one proteins were found differentially expressed in gastric cancer tissues. Of these proteins, OAT and CPS1 were found over‐expressed in gastric cancer patients, with sensitivity of 70.4% (95% CI: 63.3%–77.6%) and specificity of 80.5% (95% CI: 74.3%–86.7%) for ornithine aminotransferase, and with sensitivity of 68.6% (95% CI: 61.3%–75.8%) and specificity of 73% (95% CI: 66%–79.9%) for carbamoyl phosphate synthetase 1. The co‐expression of OAT and CPS1 in gastric cancer tissues has a sensitivity of 81% (95% CI: 73.2%–88.8%) and specificity of 89% (95% CI: 83%–95%). Furthermore, both OAT and CPS1 were overexpressed in patients with local invasion T3 and T4 stages than those in patients with T1 and T2 stages. The co‐expression of OAT and CPS1 was strongly correlated with histological grade I 68% (95% CI: 58.7%–77.3%) and TNM stage I/II 52% (95% CI: 42%–62%). The areas under ROC curves were up to 0.758 for the co‐expression of OAT and CPS1 in gastric cancer. GSEA results showed that two gene sets and 30 gene sets were activated in OAT high‐ and CPS1 high‐expression patients with gastric cancer, respectively. Conclusions The present findings indicated a tight correlation between the co‐expression of OAT and CPS1 and the histological grade, local invasion, and TNM stages of gastric cancer. Therefore, OAT and CPS1 might be predictors for gastric cancer invasion and potential targets for anticancer drug design for gastric cancer.

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Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase

Cited by 3 publications

References 50 publications

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

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

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase

Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer

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