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Enhancing human spermine synthase activity by engineered mutations.

Zhang Z, Zheng Y, Petukh M, Pegg A, Ikeguchi Y, Alexov E - PLoS Comput. Biol. (2013)

Bottom Line: It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type.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.

View Article: PubMed Central - PubMed

Affiliation: Computational Biophysics and Bioinformatics, Department of Physics, Clemson University, Clemson, South Carolina, United States of America.

ABSTRACT
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.

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Related in: MedlinePlus

Multiple sequence alignment (MSA).(A) MSA between HsSMS and TmSRM. Conserved residues are indicated by star “*”, and the four mutations are represented in bold italic letters; (B) MSA among different SMS species. The star “*” indicates the conserved residue among different species, and the residues corresponding to the mutation sites are marked in bold italic letters.
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pcbi-1002924-g001: Multiple sequence alignment (MSA).(A) MSA between HsSMS and TmSRM. Conserved residues are indicated by star “*”, and the four mutations are represented in bold italic letters; (B) MSA among different SMS species. The star “*” indicates the conserved residue among different species, and the residues corresponding to the mutation sites are marked in bold italic letters.

Mentions: The sequences of HsSMS and TmSRM were aligned at the starting point of the investigation. The alignment is shown in Figure 1A, where conserved residues are marked by star “*” and the bold italic letters indicate the four mutation sites. The query coverage is 50% and E value is 5e-13 given by Protein-BLAST [16]. As seen below, the sequences of HsSMS and TmSRM share many similarities but are also significantly different at many sites. The alignment (Figure 1A) was mapped onto HsSMS 3D structure and all sites being either invariant (with respect to amino acid substitution) or buried were removed from the candidate list. The remaining candidates, which are surface exposed residues in HsSMS (Table S1), were subjected to additional considerations, as described in supplementary material, reducing the candidate sites to twenty five. The next step was to utilize multiple sequence alignment (MSA) from different species and from homologous sequences to further reduce the list of candidates. The MSA among different species is shown in Figure 1B. The whole comparison is quite long (Supplementary materials Table S2), thus we only list the “four mutations” section in the figure. MSA of all homologous to HsSMS protein is shown in supplementary materials Table S3. The frequency of residue appearance for each of the four mutations sites is shown in Table 1.


Enhancing human spermine synthase activity by engineered mutations.

Zhang Z, Zheng Y, Petukh M, Pegg A, Ikeguchi Y, Alexov E - PLoS Comput. Biol. (2013)

Multiple sequence alignment (MSA).(A) MSA between HsSMS and TmSRM. Conserved residues are indicated by star “*”, and the four mutations are represented in bold italic letters; (B) MSA among different SMS species. The star “*” indicates the conserved residue among different species, and the residues corresponding to the mutation sites are marked in bold italic letters.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585406&req=5

pcbi-1002924-g001: Multiple sequence alignment (MSA).(A) MSA between HsSMS and TmSRM. Conserved residues are indicated by star “*”, and the four mutations are represented in bold italic letters; (B) MSA among different SMS species. The star “*” indicates the conserved residue among different species, and the residues corresponding to the mutation sites are marked in bold italic letters.
Mentions: The sequences of HsSMS and TmSRM were aligned at the starting point of the investigation. The alignment is shown in Figure 1A, where conserved residues are marked by star “*” and the bold italic letters indicate the four mutation sites. The query coverage is 50% and E value is 5e-13 given by Protein-BLAST [16]. As seen below, the sequences of HsSMS and TmSRM share many similarities but are also significantly different at many sites. The alignment (Figure 1A) was mapped onto HsSMS 3D structure and all sites being either invariant (with respect to amino acid substitution) or buried were removed from the candidate list. The remaining candidates, which are surface exposed residues in HsSMS (Table S1), were subjected to additional considerations, as described in supplementary material, reducing the candidate sites to twenty five. The next step was to utilize multiple sequence alignment (MSA) from different species and from homologous sequences to further reduce the list of candidates. The MSA among different species is shown in Figure 1B. The whole comparison is quite long (Supplementary materials Table S2), thus we only list the “four mutations” section in the figure. MSA of all homologous to HsSMS protein is shown in supplementary materials Table S3. The frequency of residue appearance for each of the four mutations sites is shown in Table 1.

Bottom Line: It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type.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.

View Article: PubMed Central - PubMed

Affiliation: Computational Biophysics and Bioinformatics, Department of Physics, Clemson University, Clemson, South Carolina, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus