title: Cloning, expression, functional validation and modeling of cinnamyl alcohol dehydrogenase isolated from xylem of Leucaena leucocephala.
abstract: A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.
authors.affiliation: Department of Biochemistry, University of Lucknow, Lucknow 226007, Uttar Pradesh, India.
authors.forename: Brijesh
authors.initials: B
authors.lastname: Pandey
authors.forename: Veda Prakash
authors.initials: VP
authors.lastname: Pandey
authors.forename: Upendra Nath
authors.initials: UN
authors.lastname: Dwivedi
mesh_terms: D000045:Acacia / Q000737:chemistry / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010636:Phenols / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids; D020005:Propanols / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014169:Transformation, Bacterial; D052584:Xylem / Q000737:chemistry / Q000201:enzymology*
chemical_list: D010636:Phenols; D010940:Plant Proteins; D020005:Propanols; D011994:Recombinant Proteins; C010559:coniferyl alcohol; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase; C020722:cinnamyl alcohol