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

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• 作者：Prashant Sonawane ; Rishi Kishore Vishwakarma ; Bashir M. Khan ; ^{bm.khan@ncl.res.in}
• 关键词：Cinnamoyl CoA reductase 1 ; SAXS ; Stability ; Activation energy ; Cinnamoyl CoA esters
• 刊名：International Journal of Biological Macromolecules
• 出版年：2013
• 出版时间：July, 2013
• 年：2013
• 卷：58
• 期：Complete
• 页码：154-159
• 全文大小：888 K

文摘

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 ¡ãC for 90 min. The enzyme showed K_cat/K_m for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (¡Á10⁶ M^?1 s^?1), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, E_a for various substrates was found to be in the range of 20-50 kJ/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 3.04 nm and volume 49.25 nm³ with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.