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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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Effects on hydrogen networks surrounding the mutation site C206.(A) WT; (B) Mutant C206R. The residues are presented in sticks: green sticks represent C206 in WT and R206 in the mutant; yellow sticks represent G238; and cyan sticks represent D239. The red dash indicates the hydrogen bonds.
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pcbi-1002924-g004: Effects on hydrogen networks surrounding the mutation site C206.(A) WT; (B) Mutant C206R. The residues are presented in sticks: green sticks represent C206 in WT and R206 in the mutant; yellow sticks represent G238; and cyan sticks represent D239. The red dash indicates the hydrogen bonds.

Mentions: The energy calculations indicated that all mutations stabilize the monomeric structure of HsSMS. The couple, E175 and H178, increases stability by lowering the desolvation penalty for D201, which is protonated in the mutant, and by providing stabilizing hydrogen bonds. The other two mutations, S165D and C206R, also stabilize the monomer, especially C206R is predicted to increase the monomer stability by 15.77 kcal/mol. In the WT structure, C206 forms a hydrogen bond with D239 (Figure 4A); while in the CRmut structure, R206 forms hydrogen bonds with the backbone oxygens of both D239 and G238 (Figure 4B). The extra hydrogen bond is the main reason for increased stability of CRmut. The S165D stabilizes the monomer as well, because the WT residue, Ser 165 is not involved in any specific interactions, while in the mutant, the Asp 165 interacts with Lys 163 and Lys 167 while being exposed, does not pay any desolvation penalty.


Enhancing human spermine synthase activity by engineered mutations.

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

Effects on hydrogen networks surrounding the mutation site C206.(A) WT; (B) Mutant C206R. The residues are presented in sticks: green sticks represent C206 in WT and R206 in the mutant; yellow sticks represent G238; and cyan sticks represent D239. The red dash indicates the hydrogen bonds.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002924-g004: Effects on hydrogen networks surrounding the mutation site C206.(A) WT; (B) Mutant C206R. The residues are presented in sticks: green sticks represent C206 in WT and R206 in the mutant; yellow sticks represent G238; and cyan sticks represent D239. The red dash indicates the hydrogen bonds.
Mentions: The energy calculations indicated that all mutations stabilize the monomeric structure of HsSMS. The couple, E175 and H178, increases stability by lowering the desolvation penalty for D201, which is protonated in the mutant, and by providing stabilizing hydrogen bonds. The other two mutations, S165D and C206R, also stabilize the monomer, especially C206R is predicted to increase the monomer stability by 15.77 kcal/mol. In the WT structure, C206 forms a hydrogen bond with D239 (Figure 4A); while in the CRmut structure, R206 forms hydrogen bonds with the backbone oxygens of both D239 and G238 (Figure 4B). The extra hydrogen bond is the main reason for increased stability of CRmut. The S165D stabilizes the monomer as well, because the WT residue, Ser 165 is not involved in any specific interactions, while in the mutant, the Asp 165 interacts with Lys 163 and Lys 167 while being exposed, does not pay any desolvation penalty.

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