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Structure of glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae at 2.45?A resolution.

ABSTRACT: The interconversion of glycerol 3-phosphate and dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenases provides a link between carbohydrate and lipid metabolism and provides Saccharomyces cerevisiae with protection against osmotic and anoxic stress. The first structure of a glycerol-3-phosphate dehydrogenase from S. cerevisiae, GPD1, is reported at 2.45?Å resolution. The asymmetric unit contains two monomers, each of which is organized with N- and C-terminal domains. The N-terminal domain contains a classic Rossmann fold with the (?-?-?-?-?)2 motif typical of many NAD+-dependent enzymes, while the C-terminal domain is mainly ?-helical. Structural and phylogenetic comparisons reveal four main structure types among the five families of glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases and reveal that the Clostridium acetobutylican protein with PDB code 3ce9 is a glycerol-1-phosphate dehydrogenase.

SUBMITTER: Alarcon DA

PROVIDER: S-EPMC3515364 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Structure of glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae at 2.45 Å resolution.

Alarcon David Aparicio DA Nandi Munmun M Carpena Xavi X Fita Ignacio I Loewen Peter C PC

Acta crystallographica. Section F, Structural biology and crystallization communications 20121026 Pt 11

The interconversion of glycerol 3-phosphate and dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenases provides a link between carbohydrate and lipid metabolism and provides Saccharomyces cerevisiae with protection against osmotic and anoxic stress. The first structure of a glycerol-3-phosphate dehydrogenase from S. cerevisiae, GPD1, is reported at 2.45 Å resolution. The asymmetric unit contains two monomers, each of which is organized with N- and C-terminal domains. The N-terminal do ...[more]

PMID: 23143232

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Project description:Autopsy-negative sudden unexplained death, including sudden infant death syndrome, can be caused by cardiac channelopathies such as Brugada syndrome (BrS). Type 1 BrS, caused by mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in the glycerol-3-phosphate dehydrogenase 1-like gene (GPD1-L) disrupted trafficking of SCN5A in a multigenerational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of sudden unexplained death/sudden infant death syndrome.Using denaturing high-performance liquid chromatography and direct DNA sequencing, we performed comprehensive open-reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 83 unrelated cases of sudden unexplained death (26 females, 57 males; average age, 14.6+/-10.7 years; range, 1 month to 48 years). A putative, sudden unexplained death-associated GPD1-L missense mutation, E83K, was discovered in a 3-month-old white boy. Further mutational analysis was then performed on genomic DNA derived from a population-based cohort of 221 anonymous cases of sudden infant death syndrome (84 females, 137 males; average age, 3+/-2 months; range, 3 days to 12 months), revealing 2 additional mutations, I124V and R273C, in a 5-week-old white girl and a 1-month-old white boy, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared with wild-type GPD1-L, GPD1-L mutations coexpressed with SCN5A in heterologous HEK cells produced a significantly reduced sodium current (P<0.01). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current (P<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS.The present study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset of sudden infant death syndrome via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal BrS-like proarrhythmic substrate.

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

Structure of glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae at 2.45?A resolution.

Publications

Structure of glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae at 2.45 Å resolution.

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