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

ChemBioChem ◽  
2019 ◽  
Vol 21 (4) ◽  
pp. 550-563 ◽  
Author(s):  
Bekir Engin Eser ◽  
Michal Poborsky ◽  
Rongrong Dai ◽  
Shigenobu Kishino ◽  
Anita Ljubic ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Rational Engineering ◽  
Critical Residues

Bioinformatics-Guided Discovery of Citrulline 4-Hydroxylase from GE81112 Biosynthesis and Rational Engineering of Its Substrate Specificity

2019 ◽  
Author(s):  
Christian Zwick ◽  
Max Sosa ◽  
Hans Renata
Keyword(s):  
Substrate Specificity ◽  
Guided Discovery ◽  
Rational Engineering

We functionally characterize a nonheme dioxygenase from GE81112 biosynthesis and identify it as a citrulline hydroxylase. A bioinformatics guided engineering was performed to alter the substrate specificity of the enzyme.

scholarly journals Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Chenghai Sun ◽  
Zhenyao Luo ◽  
Wenlu Zhang ◽  
Wenya Tian ◽  
Haidong Peng ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cytochrome P450s ◽  
Dynamics Simulation ◽  
Bioactive Natural Products ◽  
Rational Engineering ◽  
Ligand Free ◽  
Solid Foundation ◽  
Critical Residues ◽  
Mechanistic Basis ◽  
Stereo Selectivity

AbstractBacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.

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 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 Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase

2010 ◽  
Vol 23 (10) ◽  
pp. 781-787 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Bent O. Petersen ◽  
Yvonne Westphal ◽  
Adiphol Dilokpimol ◽  
Maher Abou Hachem ◽  
...  
Keyword(s):  
Lactobacillus Acidophilus ◽  
Rational Engineering ◽  
Dual Specificity ◽  
Lactobacillus Acidophilus Ncfm ◽  
Maltose Phosphorylase

N6-(Δ2-isopentenyl)adenosine: hydrolysis by a nucleosidase isolated from Lactobacillus acidophilus cells

1975 ◽  
Vol 21 (5) ◽  
pp. 633-638 ◽  
Author(s):  
J. Hordern ◽  
R. H. Johnson ◽  
B. D. McLennan
Keyword(s):  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Purine Nucleoside ◽  
Bacterial Enzyme ◽  
Electrophoretic Behavior ◽  
Isopentenyl Adenine ◽  
Isopentenyl Adenosine ◽  
Rapid Conversion

A nucleosidase activity has been isolated from Lactobacillus acidophilus which rapidly hydrolyses N6-(Δ2-isopentenyl)adenosine to its corresponding base, N6-(Δ2-isopentenyl)adenine. The activity can be distinguished from the spleen exzyme (EC. 2.4.2.1), a purine nucleoside transferase, on the basis of its substrate specificity, electrophoretic behavior, and non-dependence on phosphate. The bacterial enzyme hydrolyzes both inosine and isopentenyl adenosine, giving Km values of 63.3 μM and 177 μM respectively. The presence of this enzyme in bacteria accounts for the rapid conversion of the parent nucleoside to isopentenyl adenine, which has been observed in these cells. The enzyme thus assumes importance as one of the catabolic activities available to the cell for metabolizing the cytokinin, N6-(Δ2-isopentenyl)adenosine.

Bioinformatics-Guided Discovery of Citrulline 4-Hydroxylase from GE81112 Biosynthesis and Rational Engineering of Its Substrate Specificity

2019 ◽  
Author(s):  
Christian Zwick ◽  
Max Sosa ◽  
Hans Renata
Keyword(s):  
Substrate Specificity ◽  
Guided Discovery ◽  
Rational Engineering

We functionally characterize a nonheme dioxygenase from GE81112 biosynthesis and identify it as a citrulline hydroxylase. A bioinformatics guided engineering was performed to alter the substrate specificity of the enzyme.

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