Results for “ribokinase” | Engineering Papers

DOE OSTI2016

Identification of the Plant Ribokinase and Discovery of a Role for Arabidopsis Ribokinase in Nucleoside Metabolism

Not Available

09 BIOMASS FUELS↗

DOE OSTI2016

Crystallization and preliminary X-ray diffraction study of recombinant ribokinase from Thermus Species 2.9

Ribokinase from a thermophilic strain of Thermus species 2.9 belonging to the carbohydrate ribokinase family (EC 2.7.1.15) was isolated, purified, and crystallized. The crystallization conditions were found by the vapor-diffusion technique and were then optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals, which were grown by the counter-diffusion technique, at the SPring-8 synchrotron radiation facility to 2.87 Å resolution. The crystals belong to sp. gr. P12{sub 1}1 and have the following unit-cell parameters: a = 81.613 Å, b = 156.132 Å, c = 87.714 Å, α = γ = 90°, β = 103.819°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the protein by the molecular-replacement method.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2016

Final Report

This report summarizes our research activities. In the award period, we have made significant progress on the first aim, with new discoveries reported in one published paper (1) and in one submitted manuscript (2) currently under review. The published manuscript reports on our discovery of plant ribokinase and the metabolic pathway in which it functions; the submitted manuscript is identification and characterization of the plant fructokinase family of enzymes from expression studies, sequence comparisons, subcellular localizations and enzymatic activities of recombinant proteins. Our study of loss-of-function mutants in the fructokinase family members (2) revealed that there were no phenotypic differences observed for the five genes analyzed, so we have adopted the Crispr/Cas9 system to isolate mutants in the two genes for which there are no currently available insertion mutants, and we are generating higher order mutants (double, triples, etc) to discern the relative roles and significance for each fructokinase. These mutants will be an important resource to understand regulation of carbohydrate movement and catabolism in plants. As studies from others indicate, alteration of fructokinases results in changes in cell walls and vasculatures, which have importance relative to biofuel yield and quality. In the second aim, we have characterized the protein-protein interactions for the pkfB proteins FLN1 and FLN2 that are localized to chloroplast transcriptional complexes and have proposed a new model for how chloroplast transcription is regulated. This work has been submitted for publication, been revised and will be re-submitted in December 2016

09 BIOMASS FUELS↗

DOE OSTI2016

Studies of the PfkB Family of Proteins in Arabidopsis thaliana

The phosphofructokinase B (PkfB) family of carbohydrate kinases is a large group that includes members with specificity for a diverse array of carbohydrate substrates, including fructose, ribose, fructose‐6‐phosphate, tagatose‐6‐phosphate, adenosine, myo‐inositol, and others. Although there are 21 genes encoding PfkB family members in the higher plant Arabidopsis thaliana , only a few have been studied in detail. Two Arabidopsis proteins with fructokinase activity were identified and characterized via native PAGE and in‐gel assays, though the genes were not identified. Another class of PfkB proteins identified in Arabidopsis lacks demonstrated enzymatic activity, but is necessary for the accumulation of photosynthetic transcripts in chloroplasts. The fructokinase‐like proteins 1 and 2 (FLN1 and FLN2, respectively) and NARA5 all belong to this class, although the mechanism of how NARA5 exerts it's phenotype remains elusive. FLN1 and 2 have been identified in proteomics studies as members of a large protein complex that interacts with, and likely regulates, the prokaryotic‐type plastid RNA polymerase which specifically transcribes photosynthetic genes in the plastid. We have found that FLN1 and FLN2 interact with each other in an ATP‐dependent manner both in vitro and in vivo . Phenotypic analysis suggests that the presence of both proteins is necessary for proper levels of transcription. Our data on the interaction between FLN1 and FLN2 suggests a mechanism where the FLN proteins act as a biochemical switch to activate transcription under conditions that favor their binding to each other. We have also begun characterization of the remainder of the Arabidopsis PfkB family and have identified a group of seven highly similar Arabidopsis PfkB proteins. These seven proteins were expressed in E. coli and enzymatic assays performed. All are active as fructokinases with high specificity for fructose. T‐DNA mediated inactivation of five of the seven genes resulted in no overt phenotype which hints at possible redundancy between some of these gene products. Using GFP fusions, we have determined that all localize to the cytosol except one, which localizes to plastids. Higher order mutants and biochemical analysis, along with expression analysis will allow us to tease apart the differences and the importance of each of these family members. We have characterized another PfkB protein that appears to be the sole Arabidopsis ribokinase (AtRBSK). Its biochemical properties and cofactor requirements are similar to that of known mammalian and prokaryotic RBSKs. T‐DNA insertion into AtRBSK results in the hyper‐accumulation of ribose in both the roots and leaves of 10 day‐old seedlings compared to wild type. Arabidopsis lacking AtRBSK grow poorly on media supplemented with ribose as the only sugar. We are currently dissecting the pathway that leads to the production of ribose in plants to put AtRBSK into a biological context. Our studies of the PfkB family in Arabidopsis are yielding promising results and will continue to further our understanding of many fundamental processes in plant biology ranging from central metabolism to the regulation of transcription in chloroplasts. Support or Funding Information The authors gratefully acknowledge support from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US. Department of Energy (contracts DE‐FG02–09ER16077 and DE‐SC0002175) to JC

Riggs, John W.↗