Analysis of the Enzymatic Properties of a Broad Family of Alanine Aminotransferases

Abstract

Alanine aminotransferase (AlaAT) has been studied in a variety of organisms due to the involvement of this enzyme in mammalian processes such as non-alcoholic hepatocellular damage, and in plant processes such as C4 photosynthesis, post-hypoxic stress response and nitrogen use efficiency. To date, very few studies have made direct comparisons of AlaAT enzymes and fewer still have made direct comparisons of this enzyme across a broad spectrum of organisms. In this study we present a direct kinetic comparison of glutamate:pyruvate aminotransferase (GPAT) activity for seven AlaATs and two glutamate:glyoxylate aminotransferases (GGAT), measuring the KM values for the enzymes analyzed. We also demonstrate that recombinant expression of AlaAT enzymes in Eschericia coli results in differences in bacterial growth inhibition, supporting previous reports of AlaAT possessing bactericidal properties, attributed to lipopolysaccharide endotoxin recognition and binding. A probable lipopolysaccharide binding region within the AlaAT enzymes, homologous to a region of a lipopolysaccharide binding protein (LBP) in humans, was also identified in this study. The AlaAT enzyme differences identified here indicate that AlaAT homologues have differentiated significantly and the roles these homologues play in vivo may also have diverged significantly. Specifically, the differing kinetics of AlaAT enzymes and how this may alter the nitrogen use efficiency in plants is discussed.

Assessing the Metabolic Impact of Nitrogen Availability Using a Compartmentalized Maize Leaf Genome-Scale Model

“…This modification is predicted to compensate for the lack of GS1-4 by using the Glu and pyruvate derived from glutathione to produce Ala. Thus, it will be interesting to determine whether the increase in Ala is related to the importance of the enzyme Ala aminotransferase in the improvement of plant productivity in general and NUE in particular (Good and Beatty, 2011;McAllister et al, 2013). In all N background conditions (i.e.…”

The Arabidopsis thaliana gene annotated by the locus tag At3g08860 encodes alanine aminotransferase

“…Aminotransferase enzymes function via a bimolecular double displacement ping‐pong mechanism where an amino acid usually serves as the amino donor and an α‐keto acid serves as the amino acceptor (Nelson & Cox, ). Aminotransferases are ubiquitous in the three domains of life and are involved in a variety of metabolic pathways including amino acid metabolism, nitrogen assimilation, gluconeogenesis, responses to a number of biotic/abiotic stresses, and among other pathways (Liepman & Olsen, ; McAllister, Facette, Holt, & Good, ; Rocha et al, ; de Sousa & Sodek, ).…”

CRISPR screens in physiologic medium reveal conditionally essential genes in human cells

“…Since the two human GPTs share just 67% sequence homology, we then considered if the kinetic parameters for GPT-catalyzed formation of alanine and aKG differed between the two isoforms. However, there is little biochemical characterization reported for the human GPTs, and standard GPT assays are based on indirect readouts that rely on coupled activities (Glinghammar et al, 2009;Gubern et al, 1990;McAllister et al, 2013;Ouyang et al, 2016). To address this, we developed a new GPT activity assay in which reactions containing recombinant GPT, a pyridoxal phosphate cofactor, and the substrates pyruvate and glutamate, were evaluated by using liquid chromatography-mass spectrometry (LC-MS)-based detection of aKG ( Figures 4G and S6E).…”