TY - JOUR
T1 - Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala
AU - Sonawane, Prashant
AU - Vishwakarma, Rishi Kishore
AU - Khan, Bashir M.
N1 - Funding Information:
Authors are thankful to CSIR, Council of Scientific and Industrial Research, India for the financial support. Thanks to Ms. Pooja Singh and Mr. Manojkumar Sharma for performing DLS and SAXS experiments respectively. Authors are also thankful to Dr. Sushama Gaikwad and Dr. Guruswamy Kumarswamy for helping in data analysis and interpretations.
PY - 2013/7
Y1 - 2013/7
N2 - 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.
AB - 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.
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U2 - 10.1016/j.ijbiomac.2013.03.050
DO - 10.1016/j.ijbiomac.2013.03.050
M3 - Article
C2 - 23541561
AN - SCOPUS:84877141992
SN - 0141-8130
VL - 58
SP - 154
EP - 159
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -