An evolutionary analysis identifies a conserved pentapeptide stretch containing the two essential lysine residues for rice L-<i>myo</i>-inositol 1-phosphate synthase catalytic activity

<div><p>A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-<i>myo</i>-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, <i>Oryza sativa</i> (<i>Os</i>MIPS1). Following this study, <i>Os</i>MIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity.</p></div

Comparison of enzyme activity of wild <i>Os</i>MIPS protein and its mutants.

<p>(A) Residual MIPS activity of deletion mutants with respect to the wild type enzyme <i>Os</i>MIPS. 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 <i>Os</i>MIPS. (C) Km and (D) Vmax values for the substrate, G6P and cofactor, NAD, as calculated from Lineweaver-Burk plot.</p

Construction of <i>Os</i>MIPS deletion/substitution mutants.

<p>(A) Sequence alignment comparison study of <i>Os</i>MIPS, <i>Sc</i>MIPS and <i>Af</i>MIPS through MultAlin. Yellow arrow signifies the NAD-binding residues; red oval signifies the substrate binding residues in <i>Sc</i>MIPS; and black triangle active site residues in <i>Af</i>MIPS. Numbering of amino acids presented at the bottom line is with respect to <i>Sc</i>MIPS sequence and numbering of amino acids presented at the top line is with respect to <i>Af</i>MIPS sequence. (B) Diagrammatic representation of the generated site-specific deletion mutants of <i>Os</i>MIPS. Black arrows point to the amino acid residue (in red font) has been deleted and/or replaced by alanine from the wild-type <i>Os</i>MIPS 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.</p

Evolutionary relationships of taxa for all 172 MIPS sequences.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185351#pone.0185351.ref032" target="_blank">32</a>]. 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185351#pone.0185351.ref033" target="_blank">33</a>] 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185351#pone.0185351.ref018" target="_blank">18</a>].</p

Primers designed for <i>Os</i>MIPS mutations where each of the mentioned amino acid residues were either deleted or replaced by alanine.

<p>Primers designed for <i>Os</i>MIPS mutations where each of the mentioned amino acid residues were either deleted or replaced by alanine.</p

Conserved large stretches and blocks found among the MIPS homologous sequences.

<p>Conserved large stretches and blocks found among the MIPS homologous sequences.</p

Invariant amino acids found within the highly conserved patterns in eukaryotes and comparing them with the established crystal structures.

<p>Invariant amino acids found within the highly conserved patterns in eukaryotes and comparing them with the established crystal structures.</p

Homology stretches based on similarity percentage.

<p>(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.</p