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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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Interaction networks among product SPM, active sites: D201 & D276, and pair mutation sites.(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.
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pcbi-1002924-g003: Interaction networks among product SPM, active sites: D201 & D276, and pair mutation sites.(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.

Mentions: The experimental data shows that the designed HsSMS mutant is more active than the WT HsSMS and computational investigations indicate that biophysical characteristics of the mutant are altered as compared with the WT characteristics. What are the structural origins for the predicted changes? The MSA among homologous proteins implied that the sites 175 and 178 are correlated. In the WT HsSMS, L175 and T178 are quite close, and the polar hydrogen of T178 makes a hydrogen bond (hydrogen bond length 1.81 Å) with OD2 of Asp 201 (Figure 3A), and thus providing support for the ionized form of Asp 201. The substitution with a charged residue Glu at site 175 introduces extra negative charge, but the side chain of E175 is almost fully exposed to the water phase and thus is solvated. However, the substitution causes slight backbone reengagement and at the same time, the negative potential of E175 suppresses the ionized form of D201 and makes D201 protonated. There is no hydrogen acceptor in the vicinity of D201 and Thr at position 178 is replaced with hydrogen donor/acceptor with a longer side chain, His, to make a hydrogen bond with the polar hydrogen of D201 (Figure 3B). The negative charge of E175 also supports the tautameric orientation of the side chain of His at position 178 by orienting the polar proton of H178 toward the negatively charged OE atoms of E175 (Figure 3B). It can be speculated that by favoring the protonated form of D201, the mutations weaken the interactions between the product, the spermine, and the protein moiety and thus facilitates the release of the product (note that the product, the spermine, has one extra positive charge as compared with the reactant, the spermidine). In addition, the protonation of D201 lowers a bit the pKa value of D276 (from pKa(WT) = 9.7 to pKa(Fmut) = 9.4) and thus reduces the work need to be done to protonate D276 upon substrate binding. It can be speculated that it will enhance the turnover of the reaction and will increase the reaction rate.


Enhancing human spermine synthase activity by engineered mutations.

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

Interaction networks among product SPM, active sites: D201 & D276, and pair mutation sites.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002924-g003: Interaction networks among product SPM, active sites: D201 & D276, and pair mutation sites.(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.
Mentions: The experimental data shows that the designed HsSMS mutant is more active than the WT HsSMS and computational investigations indicate that biophysical characteristics of the mutant are altered as compared with the WT characteristics. What are the structural origins for the predicted changes? The MSA among homologous proteins implied that the sites 175 and 178 are correlated. In the WT HsSMS, L175 and T178 are quite close, and the polar hydrogen of T178 makes a hydrogen bond (hydrogen bond length 1.81 Å) with OD2 of Asp 201 (Figure 3A), and thus providing support for the ionized form of Asp 201. The substitution with a charged residue Glu at site 175 introduces extra negative charge, but the side chain of E175 is almost fully exposed to the water phase and thus is solvated. However, the substitution causes slight backbone reengagement and at the same time, the negative potential of E175 suppresses the ionized form of D201 and makes D201 protonated. There is no hydrogen acceptor in the vicinity of D201 and Thr at position 178 is replaced with hydrogen donor/acceptor with a longer side chain, His, to make a hydrogen bond with the polar hydrogen of D201 (Figure 3B). The negative charge of E175 also supports the tautameric orientation of the side chain of His at position 178 by orienting the polar proton of H178 toward the negatively charged OE atoms of E175 (Figure 3B). It can be speculated that by favoring the protonated form of D201, the mutations weaken the interactions between the product, the spermine, and the protein moiety and thus facilitates the release of the product (note that the product, the spermine, has one extra positive charge as compared with the reactant, the spermidine). In addition, the protonation of D201 lowers a bit the pKa value of D276 (from pKa(WT) = 9.7 to pKa(Fmut) = 9.4) and thus reduces the work need to be done to protonate D276 upon substrate binding. It can be speculated that it will enhance the turnover of the reaction and will increase the reaction rate.

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