Computational protocol: Common and Distant Structural Characteristics of Feruloyl Esterase Families from Aspergillus oryzae

Similar protocols

Protocol publication

[…] Primary structure analysis of the FAE amino acid sequences was performed using ProtParam, by computing various physical and chemical parameters including the molecular weight, theoretical pI, extinction coefficient and absorbance for a user entered sequence . The amino acid sequences were analyzed for the presence of native signal peptide using the SignalP 3.0 server, which predicts the presence and location of signal peptide cleavage sites in amino acid sequences based on a combination of several artificial neural networks and hidden Markov models –. The summary of calculated physico-chemical parameters and post-translational modification analysis of the mature FAEs are given in . Three-dimensional atomic models for the A. oryzae feruloylome sequences were modeled from multiple threading alignments and iterative structural assembly simulations using I-TASSER algorithm –. Structure refinement of modeled structures was carried out using the Discovery Studio software suite version 3.0 (Accelrys Inc, USA). The Prepare Protein protocol package in Discovery Studio suite was used for inserting missing atoms in incomplete residues, modeling missing loop regions , deleting alternate conformations (disorder), standardizing atom names, and protonating titratable residues using predicted pKs . The Side-Chain Refinement protocol was used for each structure to optimize the protein side-chain conformation based on systematic searching of side-chain conformation and CHARMM Polar H energy minimization using the ChiRotor algorithm . Smart Minimizer algorithm was used for the minimization process which performs 1000 steps of Steepest Descent with a RMS gradient tolerance of 3, followed by Conjugate Gradient minimization for faster convergence towards a local minimum . Structure evaluations were carried out using DOPE, which is an atomic based statistical potential in MODELER package for model evaluation and structure prediction . Structure verifications were carried out using VerifyProtein-Profiles-3D that allows evaluating the fitness of a protein sequence in its current 3D environment . [...] The RNA isolated from Aspergillus oryzae as described earlier was a kind gift from Department of Systems Biology-DTU. DNase treatment of the RNA sample and further RNA cleanup were performed according to the manufacturer’s instructions using RNase-Free DNase Set and RNeasy Mini Kit, respectively (QIAGEN Nordic, Sweden). Quality and quantity of the RNA samples were determined by using a BioPhotometer (Eppendorf, Germany). The purified RNA was stored at −80°C until further processing. The primers were designed to amplify the mature-protein coding sequences excluding the signal peptide. cDNA synthesis was carried out using the Transcriptor One-Step RT-PCR Enzyme Mix (Roche Diagnostics GmbH, Germany). Purification of the cDNA from the RT-PCR product mixture was performed using the QIAquick PCR Purification Kit according to the manufacturer’s protocol (QIAGEN Nordic, Sweden). Primers were designed using the OligoCalc tool and Clone Manager software version 9 (Scientific & Educational Software, USA). The list of oligo nucleotide primers used is given in the . The plasmid pPICZα-C vector (Invitrogen, UK) was used for cloning and expression in Pichia pastoris. [...] The assay was based on a previously reported spectrophotometric method for determination of FAE activity recommended by Biocatalysts Limited, Wales, UK , . The absorption spectra (FLUOstar Omega, BMG LABTECH, Germany) of the methyl esters of cinnamic acids and their hydrolysis products was monitored at 240–600 nm and their absorption maxima were determined . FAE activity was expressed in milliUnits (mU); 1 mU was equal to 1 nmol of ferulic acid or respective cinnamic acid released in 1 ml of the reaction medium after 1 min of incubation . The assays were done in triplicates and Km values were calculated using SigmaPlot for Windows, Version 12.0 (Systat Software Inc, USA).The expressed recombinant FAEs were tested against 15 methyl cinnamate esters (obtained from Aapin Chemicals, UK) viz., Methyl ferulate or Methyl 4-hydroxy-3-methoxy cinnamate (MFA), Methyl caffeate or Methyl 3,4-dihydroxy cinnamate (MCA), Methyl p-coumarate or Methyl 4-hydroxy cinnamate (MPC), Methyl sinapate or Methyl 4-hydroxy-3,5-dimethoxy cinnamate (MSA), Methyl 2-hydroxy cinnamate (M2C), Methyl 3-hydroxy cinnamate (M3C), Methyl cinnamate (MC), Methyl 3,4,5-trimethoxy cinnamate (MTM), Methyl 2-methoxy cinnamate (M2M), Methyl 3-methoxy cinnamate (M3M), Methyl 4-methoxy cinnamate (M4M), Methyl 3,4-dimethoxy cinnamate (M34DC), Methyl 3,5-dimethoxy cinnamate (M35DC), Methyl 3-hydroxy-4-methoxy cinnamate (M34MC) and Methyl 4-hydroxy-3-methoxy phenyl propionate (M43PP). [...] The far-UV Circular Dichroism (CD) measurements were carried on a Chirascan™ CD Spectrometer equipped with a thermostated cell holder (Applied Photophysics Limited, UK). Chirascan™ CD Spectrometer is endowed with photon flux for a 1 nm bandwidth in excess of 1013 per second for all UV wavelengths from 360 nm to 180 nm. During CD spectroscopy analysis, respective purified 6×His N-terminally tagged recombinant proteins (0.5 mg/mL) were resuspended in the corresponding buffer (pH 3.0–pH 11.0) and analysed at room temperature (∼25°C). UV CD spectra between 185 and 250 nm were collected with a data pitch of 0.1 nm, bandwidth of 2.0 nm and scanning speed of 50 nm/min . Each sample was measured in triplicate, and data between 190–240 nm were analyzed using the K2D2 method. K2D2 method uses a self-organized map of spectra from proteins with known structure to deduce a map of protein secondary structure that is used to develop the predictions –. […]

Pipeline specifications

Software tools SigmaPlot, K2D
Applications Miscellaneous, Circular dichroism spectroscopy
Organisms Aspergillus oryzae, Komagataella pastoris