scholarly journals Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

1997 ◽  
Vol 272 (4) ◽  
pp. 2218-2222 ◽  
Author(s):  
Masayuki Nakanishi ◽  
Kazuya Matsuura ◽  
Hiroyuki Kaibe ◽  
Nobutada Tanaka ◽  
Takamasa Nonaka ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Carbonyl Reductase ◽  
Mouse Lung ◽  
Coenzyme Specificity

Crystallization of Mouse Lung Carbonyl Reductase Complexed with NADPH and Analysis of Symmetry of Its Tetrameric Molecule

1995 ◽  
Vol 118 (5) ◽  
pp. 871-873 ◽  
Author(s):  
N. Tanaka ◽  
T. Nonaka ◽  
M. Nakanishi ◽  
Y. Deyashiki ◽  
A. Hara ◽  
...  
Keyword(s):  
Carbonyl Reductase ◽  
Mouse Lung

Ultrastructural localization of carbonyl reductase in mouse lung

1994 ◽  
Vol 26 (4) ◽  
pp. 311-316 ◽  
Author(s):  
Kazuya Matsuura ◽  
Yasuo Bunai ◽  
Isao Ohya ◽  
Akira Hara ◽  
Masayuki Nakanishi ◽  
...  
Keyword(s):  
Ultrastructural Localization ◽  
Carbonyl Reductase ◽  
Mouse Lung

The first crystal structure of NAD-dependent 3-dehydro-2-deoxy-D-gluconate dehydrogenase fromThermus thermophilusHB8

2014 ◽  
Vol 70 (4) ◽  
pp. 994-1004 ◽  
Author(s):  
Kudigana J. Pampa ◽  
Neratur K. Lokanath ◽  
Naoki Kunishima ◽  
Ravishankar Vittal Rai
Keyword(s):  
Crystal Structure ◽  
Hydrophobic Interactions ◽  
Thermus Thermophilus ◽  
Structural Data ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
Mouse Lung ◽  
Oligomeric State ◽  
Coenzyme Specificity ◽  
Significant Difference

2-Keto-3-deoxygluconate (KDG) is one of the important intermediates in pectin metabolism. An enzyme involved in this pathway, 3-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (DDGDH), has been identified which converts 2,5-diketo-3-deoxygluconate to KDG. The enzyme is a member of the short-chain dehydrogenase (SDR) family. To gain insight into the function of this enzyme at the molecular level, the first crystal structure of DDGDH fromThermus thermophilusHB8 has been determined in the apo form, as well as in complexes with the cofactor and with citrate, by X-ray diffraction methods. The crystal structures reveal a tight tetrameric oligomerization. The secondary-structural elements and catalytically important residues of the enzyme were highly conserved amongst the proteins of the NAD(P)-dependent SDR family. The DDGDH protomer contains a dinucleotide-binding fold which binds the coenzyme NAD+in an intersubunit cleft; hence, the observed oligomeric state might be important for the catalytic function. This enzyme prefers NAD(H) rather than NADP(H) as the physiological cofactor. A structural comparison of DDGDH with mouse lung carbonyl reductase suggests that a significant difference in the α–loop–α region of this enzyme is associated with the coenzyme specificity. The structural data allow a detailed understanding of the functional role of the conserved catalytic triad (Ser129–Tyr144–Lys148) in cofactor and substrate recognition, thus providing substantial insights into DDGDH catalysis. From analysis of the three-dimensional structure, intersubunit hydrophobic interactions were found to be important for enzyme oligomerization and thermostability.

scholarly journals Ser67Asp and His68Asp Substitutions in Candida parapsilosis Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction

2009 ◽  
Vol 75 (7) ◽  
pp. 2176-2183 ◽  
Author(s):  
Rongzhen Zhang ◽  
Yan Xu ◽  
Ying Sun ◽  
Wenchi Zhang ◽  
Rong Xiao
Keyword(s):  
Candida Parapsilosis ◽  
Enzyme Stability ◽  
Optical Purity ◽  
Carbonyl Reductase ◽  
Site Directed Mutagenesis ◽  
Short Chain ◽  
Significant Shift ◽  
Wild Type ◽  
Ketone Reduction ◽  
Coenzyme Specificity

ABSTRACT A short-chain carbonyl reductase (SCR) from Candida parapsilosis catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED) and exhibits coenzyme specificity for NADPH over NADH. By using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket. All mutations caused a significant shift of enantioselectivity toward the (R)-configuration during 2-hydroxyacetophenone reduction. The S67D/H68D mutant produced (R)-PED with high optical purity and yield in the NADH-linked reaction. By kinetic analysis, the S67D/H68D mutant resulted in a nearly 10-fold increase and a 20-fold decrease in the k cat/Km value when NADH and NADPH were used as the cofactors, respectively, but maintaining a k cat value essentially the same with respect to wild-type SCR. The ratio of Kd (dissociation constant) values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates that the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited a secondary structure and melting temperature similar to the wild-type form. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability. This work provides a protein engineering approach to modify the coenzyme specificity and enantioselectivity of ketone reduction for short-chain reductases.

Highly Active Mutants of Carbonyl Reductase S1 with Inverted Coenzyme Specificity and Production of Optically Active Alcohols

2005 ◽  
Vol 69 (3) ◽  
pp. 544-552 ◽  
Author(s):  
Souichi MORIKAWA ◽  
Takahisa NAKAI ◽  
Yoshihiko YASOHARA ◽  
Hirokazu NANBA ◽  
Noriyuki KIZAKI ◽  
...  
Keyword(s):  
Carbonyl Reductase ◽  
Optically Active ◽  
Coenzyme Specificity ◽  
Highly Active
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