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Crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae.

ABSTRACT: Nitroreductases are flavoenzymes that catalyze nitrocompounds and are widely utilized in industrial applications due to their detoxification potential and activation of biomedicinal prodrugs. Type I nitroreductases are classified into subgroups depending on the use of NADPH or NADH as the electron donor. Here, we report the crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae, one of the uncharacterized subgroups of proteins, to reveal its minimal architecture previously observed in bacterial nitroreductases such as CinD and YdjA. The structure lacks protruding helical motifs that form part of the cofactor and substrate binding site, resulting in an open and wide active site geometry. Arg82 is uniquely conserved in proximity to the substrate binding site in Frm2 homologues and plays a crucial role in the activity of the active site. Frm2 primarily utilizes NADH to reduce 4-NQO. Because missing helical elements are involved in the direct binding to the NAD(P)H in group A or group B in Type I family, Frm2 and its homologues may represent a distinctive subgroup with an altered binding mode for the reducing compound. This result provides a structural basis for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.

SUBMITTER: Song HN

PROVIDER: S-EPMC4500314 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae.

Song Hyung-Nam HN Jeong Dae-Gwin DG Bang Seo-Young SY Paek Se-Hwan SH Park Byoung-Chul BC Park Sung-Goo SG Woo Eui-Jeon EJ

Protein science : a publication of the Protein Society 20150501 7

Nitroreductases are flavoenzymes that catalyze nitrocompounds and are widely utilized in industrial applications due to their detoxification potential and activation of biomedicinal prodrugs. Type I nitroreductases are classified into subgroups depending on the use of NADPH or NADH as the electron donor. Here, we report the crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae, one of the uncharacterized subgroups of proteins, to reveal its minimal architecture previo ...[more]

PMID: 25864423

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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 the fungal nitroreductase Frm2 from Saccharomyces cerevisiae.

Publications

Crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae.

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