Cloning and in silico characterization of cinnamyl alcohol dehydrogenase gene involved in lignification of Tall fescue (Festuca arundinacea Schreb.)

Abstract

Tall fescue, a promising temperate forage grass of Himalayan region, possesses extraordinary property of rapid growth with high biomass production, but its poor digestibility due to higher lignin content limits its utilization in livestock feeding. The lignification in Tall fescue is under the control of enzymatic cascade of different regulatory enzymes. Cinnamyl alcohol dehydrogenase (CAD) is a crucial regulatory enzyme that catalyzes the last step of monolignol biosynthesis and is a potential candidate for altering the content and types of lignin, and hence increasing the digestibility of fodder crops. Hence, the present investigation was conducted on isolation, cloning and characterization of CAD gene from Tall fescue. Isolation and amplification of CAD gene resulted in an amplicon of 1521 bp. The CAD gene sequence was submitted to NCBI database with an accession number MW442831. Translation of the CAD gene sequence exhibited an ORF of 361 amino acids. The deduced CAD protein was predicted to be hydrophobic, acidic and thermally stable with molecular formula C₁₇₁₂H₂₇₃₄N₄₆₀O₅₂₀S₂₃, molecular mass of 38.82 kDa, theoretical pI of 5.60 and 3 strong transmembrane helices. The CAD protein was predicted to have a dimer forming behavior with putative NAD(P) binding site between amino acids 48 and 301, putative substrate-binding site between amino acids 48 and 301, catalytic zinc-binding site between amino acids 48 and 164 and structural zinc-binding site between amino acid residue 101 and 115. A conserved ¹⁸⁹GLGGVG¹⁹⁴ motif is the binding site for NADP(H). The conserved motif pattern of CAD's zinc catalytic center was found to be ⁶⁹GHEVVGEV(X)EVG(X)2V⁸³. The zinc-binding site was found to be conserved between amino acid 89 and 115 and was found to be ⁸⁹G(X)2VG(X)G(X)2VGXC(X)2C(X)2C(X)5QYC¹¹⁵. The deciphered sequence and putative protein information might be useful in subsequent research in lignin bioengineering for enhanced digestibility, biomass conversion as well as impact of lignin on cell wall mechanics.

Keywords: Cinnamyl alcohol dehydrogenase; Cloning; Festuca arundinacea; Lignin; Sequence analysis, protein model.