Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.Crystal structures of apo malate dehydrogenase (MDH) from Methylobacterium extorquens, MDH bound to NAD+, and MDH with oxaloacetate and ADP-ribose revealed conformational changes, closing the active site upon coenzyme and substrate binding. In the ternary complex, His284 is in position to donate a proton in the formation of (2S)-malate.Fil: Gonzalez, Javier Marcelo. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Marti Arbona, R.. Bioscience Division, Los Alamos National Laboratory; Estados UnidosFil: Chen, J. C. H.. Bioscience Division, Los Alamos National Laboratory; Estados UnidosFil: Broom Peltz, B.. Bioscience Division, Los Alamos National Laboratory; Estados UnidosFil: Unkefer, C. J.. Bioscience Division, Los Alamos National Laboratory; Estados Unido

The structure of Synechococcus elongatus enolase reveals key aspects of phosphoenolpyruvate binding

A structure-function characterization of Synechococcus elongatus enolase (SeEN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray crystal structures of SeEN in its apoenzyme form and in complex with phosphoenolpyruvate are reported at 2.05 and 2.30 Å resolution, respectively. SeEN displays the typical fold of enolases, with a conformationally flexible loop that closes the active site upon substrate binding, assisted by two metal ions that stabilize the negatively charged groups. The enzyme exhibits a catalytic efficiency of 1.2 × 105 M -1s-1for the dehydration of 2-phospho-d-glycerate, which is comparable to the kinetic parameters of related enzymes. These results expand the understanding of the biophysical features of these enzymes, broadening the toolbox for metabolic engineering applications.Fil: Gonzalez, Javier Marcelo. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Martí Arbona, Ricardo. Los Alamos National High Magnetic Field Laboratory; Estados UnidosFil: Chen, Julian C. H.. Los Alamos National High Magnetic Field Laboratory; Estados UnidosFil: Unkefer, Clifford. Los Alamos National High Magnetic Field Laboratory; Estados Unido

Producing Glucose 6-Phosphate from Cellulosic Biomass Structural Insights into Levoglucosan Bioconversion

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-D-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production

Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts

In 2010,when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortiumbegan, littlewas known about themolecular basis of algal biomass or oil production. Very fewalgal genome sequenceswere available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy\u27s Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played bymetabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels

Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography

The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s

Structure of Methylobacterium extorquens malyl-CoA lyase: CoA-substrate binding correlates with domain shift

Malyl CoA lyase (MCL) is an Mg 2+ dependent enzyme that catalyzes the reversible cleavage of (2S) 4 malyl CoA to yield acetyl CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase like family and are involved in the assimilation of one and two carbon compounds. Here, the 1.56 Å resolution X ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg 2+ is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl CoA lyase complexed with Mg 2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C terminal domain by about 30° relative to the rest of the molecule. This domain motion positions a conserved aspartate residue located in the C terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.Fil: Gonzalez, Javier Marcelo. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Marti Arbona, Ricardo. Los Alamos National Laboratory; Estados UnidosFil: Chen, Julian C. H.. Los Alamos National Laboratory; Estados UnidosFil: Unkefer, Clifford J.. Los Alamos National Laboratory; Estados Unido