@article { ISI:000320746200022,
	title = {Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala},
	journal = {International Journal of Biological Macromolecules},
	volume = {58},
	year = {2013},
	month = {JUL},
	pages = {154-159},
	publisher = {ELSEVIER SCIENCE BV},
	address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS},
	abstract = {<p>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 degrees C 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 (x10(6) 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 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. (C) 2013 Elsevier B.V. All rights reserved.</p>},
	keywords = {Activation energy, Cinnamoyl CoA esters, Cinnamoyl CoA reductase 1, SAXS, stability},
	issn = {0141-8130},
	doi = {10.1016/j.ijbiomac.2013.03.050},
	author = {Sonawane, Prashant and Vishwakarma, Rishi Kishore and Khan, Bashir Mohammad}
}
@article { ISI:000324842900004,
	title = {Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala},
	journal = {International Journal of Biological Macromolecules},
	volume = {60},
	year = {2013},
	month = {SEP},
	pages = {33-38},
	publisher = {ELSEVIER SCIENCE BV},
	address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS},
	abstract = {<p>Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10\% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in K-m values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. (c) 2013 Elsevier B.V. All rights reserved.</p>},
	keywords = {Chemical modification, Cinnamoyl CoA reductase 1, Docking simulations, homology modeling, Site directed mutagenesis, Substrate protection},
	issn = {0141-8130},
	doi = {10.1016/j.ijbiomac.2013.05.005},
	author = {Sonawane, Prashant and Patel, Krunal and Vishwakarma, Rishi Kishore and Srivastava, Sameer and Singh, Somesh and Gaikwad, Sushama M. and Khan, Bashir Mohammad}
}
