Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release.

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.

Callose plug deposition patterns vary in pollen tubes of Arabidopsis thaliana ecotypes and tomato species.

BACKGROUND: The pollen grain contains the male gametophyte that extends a pollen tube that grows through female tissues in order to deliver sperm to the embryo sac for double fertilization. Growing pollen tubes form periodic callose plugs that are thought to block off the older parts of the tube and maintain the cytoplasm near the growing tip. The morphology of callose plugs and the patterns of their deposition were previously shown to vary among species, but variation within a species had not been examined. We therefore systematically examined callose plug deposition in Arabidopsis thaliana ecotypes, tested for heritability using reciprocal crosses between ecotypes that had differing deposition patterns, and investigated the relationship between callose plugs and pollen tube growth rate. We also surveyed callose plug deposition patterns in different species of tomato. RESULTS: We used in vitro grown pollen tubes of 14 different A. thaliana ecotypes and measured the distance from the pollen grain pore to the first callose plug (termed first interval). This distance varied among Arabidopsis ecotypes and in some cases even within an ecotype. Pollen tubes without a callose plug were shorter than those with a callose plug, and tubes with a callose plug near the grain were, on average, longer than those with the first callose plug farther from the grain. Variations in the first callose plug position were also observed between different species of tomato. CONCLUSIONS: We showed that the position of the first callose plug varied among Arabidopsis ecotypes and in tomato species, and that callose plug deposition patterns were heritable. These findings lay a foundation for mapping genes that regulate callose plug deposition or that determine pollen tube length or growth rate.