Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala

Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH...

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Bibliographic Details
Published in:International journal of biological macromolecules 2013-07, Vol.58, p.154-159
Main Authors: Sonawane, Prashant, Vishwakarma, Rishi Kishore, Khan, Bashir M.
Format: Article
Language:English
Subjects:
Activation energy
active sites
Aldehyde Oxidoreductases - chemistry
Amino Acid Sequence
Catalytic Domain
Cinnamoyl CoA esters
Cinnamoyl CoA reductase 1
Coenzyme A - chemistry
Detergents - chemistry
Enzyme Stability
Escherichia coli
Esters
Fabaceae - enzymology
histidine
Hydrogen-Ion Concentration
Kinetics
Leucaena leucocephala
lysine
Metals - chemistry
Molecular Sequence Data
Molecular Weight
oxidation
Plant Proteins - chemistry
Recombinant Proteins - chemistry
SAXS
Scattering, Small Angle
Stability
Substrate Specificity
Thermodynamics
tyrosine
X-Ray Diffraction
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Summary:Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×106M−1s−1), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20–50kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04nm and volume 49.25nm3 with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.
ISSN:0141-8130
1879-0003
DOI:10.1016/j.ijbiomac.2013.03.050