The active form of vitamin B6, pyridoxal 5’-phosphate (PLP), plays an essential role in the catalytic mechanism of various proteins, including human glutamate-oxaloacetate transaminase (hGOT1), an important enzyme in amino acid metabolism. A recent molecular and genetic study showed that the E266K, R267H, and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of hGOT1, genetically suppress a developmentally arrested Arabidopsis RUS mutant. Furthermore, CD analyses suggested that the variants exist as apo proteins and implicated a possible role of PLP in the regulation of PLP homeostasis and metabolic pathways. In this work, we assessed the stability of PLP bound to hGOT1 for the three variant and wildtype (WT) proteins using a combined 6 μs of molecular dynamics (MD) simulation. For the variants and WT in the holo form, the MD simulations reproduced the “closed-open” transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing substrate access to the R387/R293 binding motif. We also showed that formation of the dimer interface is essential for PLP affinity to the active site. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network of the phosphate binding cup, which placed the cofactor for formation of the covalent Schiff base linkage with K259 for catalysis. The amino acid substitutions at positions 266, 267, and 300 reduced the structural correlation between PLP and the protein active site and/or integrity of the dimer interface. Principal component analysis and energy decomposition clearly suggested dimer misalignment and dissociation for the three variants tested in our work. The low affinity of PLP in the hGOT1 variants observed in our computational work provided structural rationale for the possible role of vitamin B6 in regulating metabolic pathways.

L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), from Bacillus anthracis was determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.

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GLUT5 is a member of the glucose transporter (GLUT) family and channels fructose but not glucose through lipid membranes. Recent crystallographic data indicated that GLUT5 may possess a unique transport mechanism from other GLUT members; however, the details of this mechanism are unclear. 32ms equilibrium dynamics in combination with restrained MD were used to generate a conformational landscape of the sugar transport mechanism in four independent systems containing a-fructose, b-fructose, a-glucose, or b-glucose. A PMF was generated for each substrate using the MBAR method. The calculated PMF revealed two energy basins which were relatively consistent across the four sugars in this work. The first basin, and consequently the succeeding energy barrier, occurred at the flexible segment of the TM7 helix at the extracellular entrance of GLUT5. This flexible region was unresolved in recent GLUT5 crystal structures, highlighting its inherent flexibility. Here for the first time, we report a conformational landscape of GLUT5 and details of the required interactions of the TM7 loop with substrate. We demonstrated that initial entrance into the GLUT5 channel required overcoming an energy barrier associated with this loop. The second basin occurred in the GLUT5 central binding pocket, where the pivotal Q166 residue that is responsible for fructose specificity, made initial contact with incoming substrate. The barrier at this location was significantly higher for glucose compared to fructose. Moreover, we observed several other regions in the channel which showed specific favorable interactions with fructose. Such interactions could not occur with glucose substrates due to their minute but important steric and electrostatic incompatibilities with GLUT5. This work improved our understanding of the GLUT5 transport mechanism that is important for future development of therapies targeting sugar metabolism.

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