Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum

Lou, Deshuai; Wang, Yue; Tan, Jun; Zhu, Lingyu; Ji, Shunlin; Wang, Bochu

doi:10.1016/j.compbiolchem.2017.08.004

Cited by 10 publications

(8 citation statements)

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“…In this way, residues G39, R40, and R41 were identified to be responsible for binding of the phosphate group at the 2′‐position of ribose. Interestingly, the importance of arginine in the binding of NAD(P) + has been shown previously on the enantiocomplementary 7α‐HSDHs. The amino acid at position 39 was mutated to an aspartate.…”

Section: Discussionmentioning

confidence: 99%

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

2018

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Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α‐ and 7β‐HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α‐OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7β‐OH). With a view to developing robust and active biocatalysts, novel NADH‐active 7β‐HSDH species are necessary to enable a solely NAD+‐dependent redox‐neutral cascade for UDCA production. A wild‐type NADH‐dependent 7β‐HSDH from Lactobacillus spicheri (Ls7β‐HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD+‐dependent 7β‐HSDH enzyme in combination with 7α‐HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD+, resulting in high yields (>90 %) of UCA and UDCA.

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Section: Discussionmentioning

confidence: 99%

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

2018

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“…The cut-off distance for Van der Waal's interactions (calculated using the particle mesh Ewald method [44]) was 10 Å. Hydrogen covalent bonds were constrained using SHAKE [45]. This length of simulation is consistent with recent studies on engineered proteins (for example, [46][47][48][49]).…”

Section: Molecular Dynamicsmentioning

confidence: 66%

Modulation of the mobility of a key region in human galactokinase: Impacts on catalysis and stability

McAuley

Huang

Timson

2018

Bioorganic Chemistry

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Galactokinase catalyses the phosphorylation of α-d-galactose and some structurally related monosaccharides. The enzyme is of interest due to its potential as a biocatalyst for the production of sugar 1-phosphates and due to its involvement in the inherited metabolic disease type II galactosemia. It has been previously shown that a region (residues 231-245) in human galactokinase often has altered mobility when active site residues are varied. We hypothesised that the reverse may be true and that designing changes to this region might affect the functioning of the active site of the enzyme. Focussing on four residues (Leu-231, Gln-242, Glu-244 and Glu-245) we conducted molecular dynamics simulations to explore the effects of changing these residues to glycine or serine. In most cases the variations resulted in local changes to the 231-245 region and global changes to the root mean squared fluctuation (RMSF) of the protein. The four serine variants were expressed as recombinant proteins. All had altered steady state enzyme kinetic parameters with α-d-galactose as a substrate. However, these changes were generally less than ten-fold in magnitude. Changes were also observed with 2-deoxy-α-d-galactose, α-d-galactosamine and α-d-talose as substrates, including (in some cases) loss of detectable activity, suggesting that these variations can tune the specificity of the enzyme. This study demonstrates that activity and specificity of human galactokinase can be modulated by variations designed to affect active site flexibility. It is likely that this principle can be generalised to other enzymes.

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“…On the other hand, a positively charged Arg residue (R40, Figure A; highlighted in blue) on the β2α3 loop of the NADPH-dependent enzymes was also well conserved, because it forms not only an electrostatic interaction with the 2′-phosphate group of NADPH (phospho-) adenosine ribose but also a pi-cation interaction with the adenine moiety of the cofactor (Figure B). , Meanwhile, although Arg41 in Rt 7β-HSDH is not well conserved in NADPH-dependent enzymes, it can also form a favorable electrostatic interaction with the 2′-phosphate group of NADPH. As NADH-dependent enzymes prefer negatively charged residues, we introduced two other potentially favorable mutations, R40D and R41D, which would alter the coenzyme dependence according to in silico analysis.…”

Section: Resultsmentioning

confidence: 99%

Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid

et al. 2018

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Dehydrogenases are widely employed as biocatalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxidoreductases. 7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme for the biochemical synthesis of ursodeoxycholic acid (UDCA). To date, all reported 7β-HSDHs are strictly NADPH-dependent enzymes. However, compared with NADPH, NADH is much more economical, making it the preferential cofactor for synthetic applications of dehydrogenases. In this work, a recombinant 7β-HSDH originating from Ruminococcus torques was rationally engineered to alter its cofactor dependence using a strategy referred to as Cofactor Specificity Reversal: Small-and-Smart Library Design (CSR-SaSLiD), which is based on structural information and conservative sequence alignment. We rationally designed a small-and-smart library containing only five mutants that enabled the quick identification of target variants. Compared with the wild type, the resultant mutant, G39D, showed a 953 000-fold switch in cofactor specificity from NADPH to NADH, and another mutant, G39D/T17A, resulted in 223-fold enhanced activity with NADH. The structural mechanism regarding the effect of mutation on the reversal of cofactor preference and improvement of catalytic activity was elucidated with the aid of molecular dynamics simulation. Furthermore, it was confirmed that the CSR-SaSLiD strategy can be extended to other 7β-HSDHs. This work provides an efficient approach to altering cofactor preference and subsequently recovering the enzymatic activity of dehydrogenases for cost-effective biotechnical applications.

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Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum

Cited by 10 publications

References 20 publications

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

Modulation of the mobility of a key region in human galactokinase: Impacts on catalysis and stability

Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid

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Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum

Cited by 10 publications

References 20 publications

NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

NAD+‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

Modulation of the mobility of a key region in human galactokinase: Impacts on catalysis and stability

Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid

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Product

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NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids

NAD⁺‐Dependent Enzymatic Route for the Epimerization of Hydroxysteroids