Table 1.
Primers designed for OsMIPS mutations where each of the mentioned amino acid residues were either deleted or replaced by alanine.
Fig 1.
Evolutionary relationships of taxa for all 172 MIPS sequences.
Prokaryotes and Eukaryotes have a well-defined boundary. Eukaryotic sequences have been denoted by shaded zone. The evolutionary history was inferred using the Neighbor-Joining method [32]. The optimal tree with the sum of branch length = 18.55289526 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [33] and are in the units of the number of amino acid substitutions per site. The analysis involved 172 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 222 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [18].
Table 2.
Conserved large stretches and blocks found among the MIPS homologous sequences.
Fig 2.
Conserved region identified in MultAlin of all the 172 MIPS sequences selected.
Long stretches found to be phylogenetically conserved are shown in the figure. The red shaded zones within the denoted stretches represent the six phylogenetically conserved blocks present in eukaryotes. Stretch-1 consists of only block-F with two different sites of occurrence.
Fig 3.
Homology stretches based on similarity percentage.
(A) Box- plot for Similarity % of three large conserved stretches across evolution. The horizontal line within each box denotes median values, ‘+’ sign denotes the mean value. (B) Loci of Blocks A to E are compared among the selected eukaryotic MIPS sequences.
Table 3.
Invariant amino acids found within the highly conserved patterns in eukaryotes and comparing them with the established crystal structures.
Fig 4.
Construction of OsMIPS deletion/substitution mutants.
(A) Sequence alignment comparison study of OsMIPS, ScMIPS and AfMIPS through MultAlin. Yellow arrow signifies the NAD-binding residues; red oval signifies the substrate binding residues in ScMIPS; and black triangle active site residues in AfMIPS. Numbering of amino acids presented at the bottom line is with respect to ScMIPS sequence and numbering of amino acids presented at the top line is with respect to AfMIPS sequence. (B) Diagrammatic representation of the generated site-specific deletion mutants of OsMIPS. Black arrows point to the amino acid residue (in red font) has been deleted and/or replaced by alanine from the wild-type OsMIPS following the procedure described in Materials and Methods section. The box represents the “core catalytic domain” and the four evolutionarily conserved blocks within the catalytic site are underlined.
Fig 5.
Comparison of enzyme activity of wild OsMIPS protein and its mutants.
(A) Residual MIPS activity of deletion mutants with respect to the wild type enzyme OsMIPS. Black arrows indicate the four lysine deletion mutants having least enzyme activity. (B) Residual MIPS activity of deletion and replacement mutants of lysine residues within the catalytic domain represented as KΔ and K/A, respectively. ΔKEISK represents the pentapeptide (inset) deletion mutant while K355A, K359A represents double substitution at positions 355 and 359 of OsMIPS. (C) Km and (D) Vmax values for the substrate, G6P and cofactor, NAD, as calculated from Lineweaver-Burk plot.
Fig 6.
Phylogenetic conservation of the pentapeptide, K-x-x-x-K.
(A) MultAlin of the 'core catalytic domain’ among representative MIPS sequences from each phyla- Mycobacterium tuberculosis, Thermotoga maritima, Saccharomyces cerevisiae, Candida albicans, Entamoeba histolytica, Trypanosoma vivax, Oryza sativa, Arabidopsis thaliana, Caenorhabditis elegans and Homo sapiens showing 100% conservation of the two lysine residues belonging to pentapeptide. (B) Loci of the conserved lysine residues in the MIPS sequences in the established crystal structures, MtMIPS, TmMIPS AfMIPS, ScMIPS and CeMIPS compared with MIPS from Oryza sativa, (OsMIPS). As per SWISSMODEL, OsMIPS protein structure closely resembles ScMIPS, shown within black bounding box. (C) The conserved amino acids with ≥90% similarity within the four conserved blocks of catalytic domain are marked on yeast MIPS crystal structure, 1RM0 complexed with inhibitor, D6P and cofactor, NADH. The two lysine residues belonging to conserved pentapeptide are represented in red font. The figure has been generated using PyMOL. To make the residues of the catalytic core visible, some of the residues have been removed from the figure.