Enhancing Human Spermine Synthase Activity by Engineered Mutations

<div><p>Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing.</p> </div

3D structure of HsSMS dimer in ribbon presentation.

<p>Four mutation sites are shown with ball representation: Yellow: S165D; Magenta: C206R; Cyan: L175E; and Orange: T178H.</p

The results of structure – based (3C6K) folding energy calculation on monomer stability changes under three force fields.

*<p>Two parameters were used to adjust the prediction according to the experimental data. For details of sMMGB refer to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002924#pcbi.1002924-Zhang5" target="_blank">[53]</a>.</p

The results of structure – based (3C6K) binding energy calculation on dimer affinity changes under three force fields.

<p>The results of structure – based (3C6K) binding energy calculation on dimer affinity changes under three force fields.</p

pKa value of Glu175, His178 and Asp201 in C monomer of Pmut.

<p>pKa value of Glu175, His178 and Asp201 in C monomer of Pmut.</p

Comparison of SMS activity between WT and Fmut.

<p>Comparison of SMS activity between WT and Fmut.</p

Interaction networks among product SPM, active sites: D201 & D276, and pair mutation sites.

<p>(A) WT; (B) Pmut. Pair mutation sites were shown with sticks: magenta represented L175 in WT and E175 in mutant; blue represents T178 in WT and H178 in mutant. The active sites (D201 and D276) and SPM are shown in ball and sticks: yellow represents SPM; orange represents D276 and cyan represents D201. The short red lines indicate hydrogen bonds.</p

Frequency and percentage of residue appearance at the mutation sites among 500 homologous proteins.

<p>Frequency and percentage of residue appearance at the mutation sites among 500 homologous proteins.</p

Potential distribution.

<p>(A) Electrostatic field lines for the WT HsSMS; Drawing method: FieldLines; GardientMag: 1.45; Min Length: 35.31; Max Length: 50.90; Coloring Scale Data Range: −1; 1; (B) Potential difference (mutant – WT) mapped onto HsSMS surface; Coloring method: Volume; 1. Drawing method: Surf; Coloring Scale Data Range: −1; 1 (blue – positive, red – negative).</p

The results of normal mode analysis.

<p>The results of normal mode analysis.</p