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Crystal structure of Saccharomyces cerevisiae Aro8, a putative ?-aminoadipate aminotransferase.

ABSTRACT: ?-Aminoadipate aminotransferase (AAA-AT) catalyzes the amination of 2-oxoadipate to ?-aminoadipate in the fourth step of the ?-aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA-AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2-oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA-AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine-pyridoxal-5-phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate-binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA-AT/kynurenine aminotransferases.

SUBMITTER: Bulfer SL

PROVIDER: S-EPMC3795499 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Crystal structure of Saccharomyces cerevisiae Aro8, a putative α-aminoadipate aminotransferase.

Bulfer Stacie L SL Brunzelle Joseph S JS Trievel Raymond C RC

Protein science : a publication of the Protein Society 20130828 10

α-Aminoadipate aminotransferase (AAA-AT) catalyzes the amination of 2-oxoadipate to α-aminoadipate in the fourth step of the α-aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA-AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2-oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA-AT LysN from Therm ...[more]

PMID: 23893908

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Project description:BackgroundAs the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.ResultsThe crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 A resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-alpha helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 +/- 9 microM for 6-phosphogluconate and of 35 +/- 6 microM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.ConclusionThe overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.

Dataset Information

Crystal structure of Saccharomyces cerevisiae Aro8, a putative ?-aminoadipate aminotransferase.

Publications

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α-aminoadipate aminotransferase.

Similar Datasets

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