Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization.
Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents.
Division of Plant Tissue Culture, CSIR-National Chemical Laboratory, Pune 411008, India.
Parth
P
Patel
Division of Plant Tissue Culture, CSIR-National Chemical Laboratory, Pune 411008, India.
Neha
N
Gupta
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
Sushama
S
Gaikwad
Division of Plant Tissue Culture, CSIR-National Chemical Laboratory, Pune 411008, India.
Dinesh C
DC
Agrawal
Division of Plant Tissue Culture, CSIR-National Chemical Laboratory, Pune 411008, India. Electronic address: bm.khan@ncl.res.in.
Bashir M
BM
Khan
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D007887:Fabaceae / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008031:Lignin; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase
Cinnamyl alcohol dehydrogenase (CAD); Metalloenzyme; Substrate specificity