Site-Directed Mutagenesis of Residues in Coenzyme-Binding Domain and Active Site of Mouse Lung Carbonyl Reductase

Nakanishi, Mahito; Kaibe, Hiroyuki; Matsuura, Kiyotaka; Kakumoto, Mikio; Tanaka, Nobutada; Nonaka, Takamasa; Mitsui, Yukio; Hara, Akira

doi:10.1007/978-1-4615-5871-2_63

Cited by 5 publications

(5 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, the S159A mutant indicated that the K m value for 9,10-phenanthrenequinone (model substrate) was 5.5 times higher for the mutant than the wild-type enzyme (0.011 mM for S159A mutant vs 0.002 mM for the wild-type). These results indicate that the role of Ser159, Tyr178 and Lys182 residues in the Tx chicken fatty liver CR is similar to that of the residues reported for the mouse lung CR [26]. Thus, solving the structure of the Tx chicken fatty liver CR-NADPHsubstrate ternary complex could be useful for further investigation purpose.…”

supporting

confidence: 78%

A novel NAD(P)H‐dependent carbonyl reductase specifically expressed in the thyroidectomized chicken fatty liver: catalytic properties and crystal structure

et al. 2015

View full text Add to dashboard Cite

A gene encoding a functionally unknown protein that is specifically expressed in the thyroidectomized chicken fatty liver and has a predicted amino acid sequence similar to that of NAD(P)H-dependent carbonyl reductase was overexpressed in Escherichia coli; its product was purified and characterized. The expressed enzyme was an NAD(P)H-dependent broad substrate specificity carbonyl reductase and was inhibited by arachidonic acid at 1.5 lM. Enzymological characterization indicated that the enzyme could be classified as a cytosolic-type carbonyl reductase. The enzyme's 3D structure was determined using the molecular replacement method at 1.98A resolution in the presence of NADPH and ethylene glycol. The asymmetric unit consisted of two subunits, and a noncrystallographic twofold axis generated the functional dimer. The structures of the subunits, A and B, differed from each other. In subunit A, the active site contained an ethylene glycol molecule absent in subunit B. Consequently, Tyr172 in subunit A rotated by 103.7°in comparison with subunit B, which leads to active site closure in subunit A. In Y172A mutant, the K m value for 9,10-phenanthrenequinone (model substrate) was 12.5 times higher than that for the wild-type enzyme, indicating that Tyr172 plays a key role in substrate binding in this carbonyl reductase. Because the Tyr172-containing active site lid structure (Ile164-Gln174) is not conserved in all known carbonyl reductases, our results provide new insights into substrate binding of carbonyl reductase. The catalytic properties and crystal structure revealed that thyroidectomized chicken fatty liver carbonyl reductase is a novel enzyme.

show abstract

supporting

confidence: 78%

A novel NAD(P)H‐dependent carbonyl reductase specifically expressed in the thyroidectomized chicken fatty liver: catalytic properties and crystal structure

et al. 2015

View full text Add to dashboard Cite

show abstract

“…In contrast, the drastic loss of the enzyme activity by this replacement suggests that the hydroxyl side chain of Tyr-180 is important for the catalytic function of the enzyme. Although the enzyme activity was not completely inactivated by the Y180F mutation, it has been reported that rat 3␣-hydroxysteroid dehydrogenase (20) and mouse lung carbonyl reductase (21), which are characterized to have a catalytic tyrosine residue by crystallographic studies of their ternary complexes, retain low activity after the replacement of the catalytic residue with phenylalanine. The present finding, while not conclusive, is supportive evidence that the tyrosine at this position plays a critical role in the catalytic mechanism of Z. mobilis GFO as well as monkey DD.…”

Section: Resultsmentioning

confidence: 97%

Roles of His-79 and Tyr-180 of -Xylose/Dihydrodiol Dehydrogenase in Catalytic Function

Asada

Aoki

Ishikura

et al. 2000

Biochemical and Biophysical Research Communications

Self Cite

View full text Add to dashboard Cite

“…Although exchanging amino acids equal to Ala31 in Gre2p successfully changed cofactor preference from NADPH to NADH in other SDRs like mouse lung carbonyl reductase [63] and human estrogenic 17-β-hydroxysteroid dehydrogenase [38], no change in cofactor preference could be observed in Gre2p when Ala31 was replaced by Asp and Glu, respectively. Instead both enzyme variants showed a significant loss of activity regardless of the cofactor used what clearly disqualifies substitution of Ala31 as a strategy for altering cofactor preference in Gre2p.…”

Section: Resultsmentioning

confidence: 99%

Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

Katzberg

Parachin

Gorwa-Grauslund

et al. 2010

IJMS

View full text Add to dashboard Cite

The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the γ-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.

show abstract

Site-Directed Mutagenesis of Residues in Coenzyme-Binding Domain and Active Site of Mouse Lung Carbonyl Reductase

Cited by 5 publications

References 26 publications

A novel NAD(P)H‐dependent carbonyl reductase specifically expressed in the thyroidectomized chicken fatty liver: catalytic properties and crystal structure

A novel NAD(P)H‐dependent carbonyl reductase specifically expressed in the thyroidectomized chicken fatty liver: catalytic properties and crystal structure

Roles of His-79 and Tyr-180 of -Xylose/Dihydrodiol Dehydrogenase in Catalytic Function

Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

Contact Info

Product

Resources

About