scholarly journals DNA polymerase β: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics

1997 ◽  
Vol 323 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Vadim S. KRAYNOV ◽  
Brian G. WERNEBURG ◽  
Xuejun ZHONG ◽  
Hui LEE ◽  
Jinwoo AHN ◽  
...  
Keyword(s):  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Catalytic Efficiency ◽  
Main Function ◽  
Wild Type ◽  
Active Site Residues ◽  
Dna Polymerase Β ◽  
Steady State Kinetics

DNA polymerase β (pol β) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd (app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol β, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol β. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol β. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol β. Since the chemical step may not be rate-limiting for wild-type pol β, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol β displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

scholarly journals Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

2019 ◽  
Author(s):  
Bekir Engin Eser ◽  
Michal Poborsky ◽  
Rongrong Dai ◽  
Shigenobu Kishino ◽  
Anita Ljubic ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Wild Type ◽  
Active Site Residues ◽  
Diverse Range ◽  
Preparative Scale ◽  
Conversion Rates

<div>Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from <i>Lactobacillus acidophilus</i>: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards omega-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for <i>cis</i>-5, <i>cis</i>-8, <i>cis</i>-11,<i> cis</i>-14, <i>cis</i>-17-eicosapentaenoic acid (EPA) and <i>cis</i>-8, <i>cis</i>-11, <i>cis</i>-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.</div><div><br></div>

Aphidicolin-resistant and -sensitive base excision repair in wild-type and DNA polymerase β-defective mouse cells

DNA Repair ◽  
2004 ◽  
Vol 3 (7) ◽  
pp. 703-710 ◽  
Author(s):  
Eleonora Parlanti ◽  
Barbara Pascucci ◽  
Gloria Terrados ◽  
Luis Blanco ◽  
Eugenia Dogliotti
Keyword(s):  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Wild Type ◽  
Dna Polymerase Β ◽  
Mouse Cells ◽  
Base Excision

scholarly journals Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

2019 ◽  
Author(s):  
Bekir Engin Eser ◽  
Michal Poborsky ◽  
Rongrong Dai ◽  
Shigenobu Kishino ◽  
Anita Ljubic ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Wild Type ◽  
Active Site Residues ◽  
Diverse Range ◽  
Preparative Scale ◽  
Conversion Rates

<div>Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from <i>Lactobacillus acidophilus</i>: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards omega-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for <i>cis</i>-5, <i>cis</i>-8, <i>cis</i>-11,<i> cis</i>-14, <i>cis</i>-17-eicosapentaenoic acid (EPA) and <i>cis</i>-8, <i>cis</i>-11, <i>cis</i>-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.</div><div><br></div>

scholarly journals Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

1994 ◽  
Vol 301 (3) ◽  
pp. 829-834 ◽  
Author(s):  
S Daff ◽  
F D Manson ◽  
G A Reid ◽  
S K Chapman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Catalytic Efficiency ◽  
Hydroxy Acids ◽  
Wild Type ◽  
Active Site Residues ◽  
Sequence Comparisons ◽  
Flavocytochrome B2 ◽  
Strategic Manipulation ◽  
Primary Substrate

Flavocytochrome b2 from Saccharomyces cerevisiae acts physiologically as an L-lactate dehydrogenase. Although L-lactate is its primary substrate, the enzyme is also able to utilize a variety of other (S)-2-hydroxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues required for catalysis. However, the residues Ala-198 and Leu-230, found in the X-ray-crystal structure to be in contact with the substrate methyl group, are not well conserved. We propose that the interaction between these residues and a prospective substrate molecule has a significant effect on the substrate specificity of the enzyme. In an attempt to modify the specificity in favour of larger substrates, three mutant enzymes have been produced: A198G, L230A and the double mutant A198G/L230A. As a means of quantifying the overall kinetic effect of a mutation, substrate-specificity profiles were produced from steady-state experiments with (S)-2-hydroxy acids of increasing chain length, through which the catalytic efficiency of each mutant enzyme with each substrate could be compared with the corresponding wild-type efficiency. The Ala-198-->Gly mutation had little influence on substrate specificity and caused a general decrease in enzyme efficiency. However, the Leu-230-->Ala mutation caused the selectivity for 2-hydroxyoctanoate over lactate to increase by a factor of 80.

scholarly journals USP47 Is a Deubiquitylating Enzyme that Regulates Base Excision Repair by Controlling Steady-State Levels of DNA Polymerase β

2011 ◽  
Vol 41 (5) ◽  
pp. 609-615 ◽  
Author(s):  
Jason L. Parsons ◽  
Irina I. Dianova ◽  
Svetlana V. Khoronenkova ◽  
Mariola J. Edelmann ◽  
Benedikt M. Kessler ◽  
...  
Keyword(s):  
Steady State ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Polymerase Β ◽  
Steady State Levels ◽  
Base Excision

scholarly journals Rational Engineering of Hydratase from Lactobacillus Acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

2019 ◽  
Author(s):  
Bekir Engin Eser ◽  
Michal Poborsky ◽  
Rongrong Dai ◽  
Shigenobu Kishino ◽  
Anita Ljubic ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Wild Type ◽  
Active Site Residues ◽  
Diverse Range ◽  
Preparative Scale ◽  
Conversion Rates

<div>Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from Lactobacillus acidophilus: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards -end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (kcat/Km) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA) and cis-8, cis-11, cis-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.</div><div><br></div>

scholarly journals The human checkpoint sensor and alternative DNA clamp Rad9–Rad1–Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery

2005 ◽  
Vol 389 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Ekaterina SMIRNOVA ◽  
Magali TOUEILLE ◽  
Enni MARKKANEN ◽  
Ulrich HÜBSCHER
Keyword(s):  
Quality Control ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Dna Polymerase Β ◽  
Flap Endonuclease 1 ◽  
Important Target ◽  
Dna Ligase I ◽  
Base Excision

The human checkpoint sensor and alternative clamp Rad9–Rad1–Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9–Rad1–Hus1 with MutY DNA glycosylase, DNA polymerase β and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9–Rad1–Hus1 complex, thus enhancing the quality control of DNA.

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