Molecular Characterization and Expression of a Novel Alcohol Oxidase from <i>Aspergillus terreus</i> MTCC6324

<div><p>The alcohol oxidase (AOx) cDNA from <i>Aspergillus terreus</i> MTCC6324 with an open reading frame (ORF) of 2001 bp was constructed from <i>n</i>-hexadecane induced cells and expressed in <i>Escherichia coli</i> with a yield of ∼4.2 mg protein g⁻¹ wet cell. The deduced amino acid sequences of recombinant rAOx showed maximum structural homology with the chain B of aryl AOx from <i>Pleurotus eryngii</i>. A functionally active AOx was achieved by incubating the apo-AOx with flavin adenine dinucleotide (FAD) for ∼80 h at 16°C and pH 9.0. The isoelectric point and mass of the apo-AOx were found to be 6.5±0.1 and ∼74 kDa, respectively. Circular dichroism data of the rAOx confirmed its ordered structure. Docking studies with an <i>ab-initio</i> protein model demonstrated the presence of a conserved FAD binding domain with an active substrate binding site. The rAOx was specific for aryl alcohols and the order of its substrate preference was 4-methoxybenzyl alcohol >3-methoxybenzyl alcohol>3, 4-dimethoxybenzyl alcohol > benzyl alcohol. A significantly high aggregation to ∼1000 nm (diameter) and catalytic efficiency (<i>k_cat/K_m</i>) of 7829.5 min⁻¹ mM⁻¹ for 4-methoxybenzyl alcohol was also demonstrated for rAOx. The results infer the novelty of the AOx and its potential biocatalytic application.</p></div

An overall schematic diagram highlighting the proteomics and genomics approach undertaken to characterize AOx cDNA.

<p>Target protein is marked in red star. AOX 1, AOX 2 and AOX 3 represent the amino acid sequences of different AOxs. F 1, F 2, R 1 and R 2 represent the PCR primers.</p

Steady-state kinetic parameters of <i>in-vitro</i> refolded recombinant alcohol oxidase from <i>E.coli</i>.

<p>Mean <i>K</i>_m, <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m values were determined and all assays were performed in replicates of 3 (n = 3).</p

Docking view of modeled rAOx (FAD docked) with its alcohol substrates.

<p>Docking view of aromatic alcohols (highlighted as thick stick CPK model) as substrates with FAD docked (highlighted as thick stick CPK model) apo-rAOx holoenzyme complex. Conserved amino acid residues hypothesized to take part in catalytic reaction in oxidizing its substrates are highlighted as thin stick Corey-Pauling-Koltun (CPK) model with its residues labelled. Panel (<b>A</b>), (<b>B</b>), (<b>C</b>) and (<b>D</b>) shows the close-up docking view generated by Molegro Virtual Docker version 4.0.2 (CLC bio-Qiagen company) of <i>ρ</i>-methoxybenzyl alcohol; <i>m</i>-methoxybenzyl alcohol; 3,4 dimethoxybenzyl alcohol and benzyl alcohol, respectively.</p

Matching peptides of AOx corresponding to conserved internal amino acid sequence as predicted by FindPept pmf search for tryptic digest of 2-D gel spot 6.

<p>The pmf peak list (m/z values) generated from spot 6 of 2D gel was manually uploaded on FindPept online tool. Detectable m/z values were matched with the theoretical peptide masses generated from the virtual tryptic digest of query amino acid sequence of AOx from <i>A.terreus</i> NIH2624 strain as input. The output result shows user mass (practical), database mass (theoretical), delta mass (theoretical minus practical mass), peptide sequence and corresponding position in query sequence. The above table highlights only those two peptide fragments which correspond to conserved internal amino acid blocks from multiple sequence alignment of similar AOx from other filamentous fungi and yeast species.</p

I-TASSER predicted <i>ab-initio</i> model of apo-rAOx docked with its co-factor FAD.

<p>(<b>A</b>). The best model (predicted by I-TASSER) docked with FAD using Molegro Virtual Docker. FAD is represented as Corey-Pauling-Koltun (CPK) model bound at its conserved Rossmann fold motif (GXGXXG) highlighted by yellow dotted square with its residue labeled as shown as a magnified view in panel (<b>B</b>) in the model and also shown against the N-terminal loops region present between the first β-sheet (highlighted as blue bar) and first α-helix (highlighted as red bar) in the lower panel (<b>C</b>). The loop region takes part in non-covalent interaction with FAD, thus stabilizing the overall structure. Red ribbon shows the α-helices, blue ribbon depicts β-sheets and loops are shown in grey tubular wire.</p

Fluorescence spectra and Circular dichroic spectra of refolded rAOx with co-factor FAD as holoenzyme.

<p>(A). Decrease in fluorescence emission maxima of free FAD (solid black curve) and FAD reconstituted holoenzyme (rAOx) (black dashed curve) at λ_∼527 nm diluted in 20 mM sodium phosphate buffer pH 9.0 shows the quenching due to successful incorporation of FAD in the protein core during refolding after ∼80 h incubation at specified condition. (B). Dichroic spectra of rAOx from λ₁₉₀ nm to λ₂₆₀ nm in both basic and acidic environment taken after ∼80 h incubation at 4°C. Spectrum of purified apo-rAOx in identical folding buffer without glutathione and DTT was also studied as a control (red spectrum). Spectra of FAD bound rAOx fraction were recorded after removal of unbound FAD by micro-centrifugal filtration and diluting the holoenzyme rAOx in 20 mM of the respective pH buffer at a range from 9.0 to 4.0. CD spectra at pH 9.0 (pink spectrum) and pH 6.0 (black dashed spectrum) overlaps due to similar conformational stability at the respective pH environment.</p

Nucleotide and deduced amino acid sequence of AOx from <i>A.terreus</i> MTCC6324.

<p>Double stranded primer walking confirmed an ORF of 2001(−) denotes a stop codon. The N-terminal conserved amino acids taking part in Rossmann fold architecture (GXGXXG motif) are underlined in black with its residues in bold. The full length cDNA is submitted to NCBI GenBank with accession no: JX139751.</p

DLS analysis of rAOx, cAOx from <i>Pichia pastoris</i> and BSA for 0 h and 24 h incubation, respectively at 16°C.

<p>Readings were taken after removal of unbound FAD through micro-centrifugal filtration and subsequent filtering through 0.22 micron syringe filter of each time-point samples. (<b>A</b>). Onset of aggregation immediately after mixing the purified apo-rAOx with FAD in refolding buffer (0 h), the presence of peak at diameter (d) = ∼10 nm (panel <b>A1</b>), confirms the onset of aggregation. Panel <b>A2</b> shows the 0 h DLS signal of cAOx containing high aggregation reflected by a broad peak at diameter (d) = ∼1000 nm. Panel <b>A3</b> represents the aggregation profile of BSA at 0 h. (<b>B</b>). Panel <b>B1</b>, shows the highly aggregated complex formed after 24 h incubation of rAOx with major peak shift to diameter (d) = ∼1000 nm. Panel <b>B2</b> shows the constant aggregation profile of cAOx with no major peak shift when compared to 0 h data. Panel <b>B3</b> represents the constant aggregation profile of BSA, no major peak shift suggests no higher aggregated complex formed after incubation for 24 h and acted as a positive control.</p

Ligation of overlapping PCR products at common restriction site and the full length AOx cDNA clone confirmation in TA vector through restriction digestion and double stranded DNA sequencing by primer walking.

<p>(<b>A</b>). Amplified overlapping PCR amplicons using <i>A.terreus</i> MTCC 6324 cDNA as PCR template. Lane M represents wide range DNA marker, lane L1 represents PCR 1, a ∼1577 bp cDNA fragment of AOx from its start codon, lane L2 represents PCR 2, a ∼1278 bp overlapping cDNA fragment of AOx till the stop codon. Qualitative gel analysis was performed in 0.8% agarose concentration stained with ethidium bromide. (<b>B</b>). Restriction digestion pattern of ligated overlapping PCR fragments in TA vector with <i>EcoR</i>I restriction enzyme. The release of ∼2001 bp full length AOx fragment from TA vector backbone of ∼3015 bp was evident. Qualitative gel analysis was performed in 0.8% agarose concentration stained with ethidium bromide. (<b>C</b>). Pair-wise sequence alignment of primer walking DNA sequence with the un-reviewed AOx sequence information from <i>A.terreus</i> NIH strain at nucleotide and amino acid level shows a deletion of six contiguous nucleotide sequences (position 685–690) which codes for two serine residues at position 186 and 187. Corresponding sharp nucleotide chromatogram of the deleted region is highlighted, confirming the good quality of the sequencing data and ruling out any possible error in sequencing. Highly similar residues are highlighted in black. (<b>D</b>). Qualitative agarose gel for PCR amplification of full length AOx from TA vector. Lane M is a wide range DNA marker, lane L1, shows PCR amplicon of AOx at ∼20001 bp using forward and reverse primers with <i>EcoR</i>I and <i>Hind</i>III restriction sites, respectively.</p