Figure 1.
An overall schematic diagram highlighting the proteomics and genomics approach undertaken to characterize AOx cDNA.
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.
Figure 2.
2D electrophoresis of microsomal membrane bound proteins and multiple sequence alignment of highly similar AOx proteins from filamentous fungi and yeast species showing the primers for overlapping PCR based on internal peptide fragments identified from pmf data of spot 6.
(A). Mass spectrometry compatible silver stained 2D gel image of A.terreus MTCC6324 microsomal proteome. 150 µg of A.terreus microsomal protein having high AOx activity were resolved on 3–10 linear immobiline dry pH gradient strip as first dimension and subsequently run on 12% polyacrylamide resolving gel as second dimension. Seven spots (marked 1–7 along with arrow heads) showing the highest normalized volumes were analysed using MALDI-TOF-MS. (B). Internal peptide fragment 1 (GVATVPSKP) and fragment 2(NHITAGIQHGWSHP) shown in red bars, served as templates for designing overlapping PCR primers mapped as AOX-FP2 and AOX-RP1, respectively as an approach for characterizing full length AOx coding region. The gene identification numbers (GI) for the aligned amino acid sequences of AOxs are as follows: P.angusta (GI:113652); C.boidinii (GI:231528); A.terreus NIH2624 (GI:115437438); K.pastoris (GI: 2104963); C. Victoriae (GI: 13182929); P.fulva (GI: 9082281); P.chrysogenum (GI: 18028450). Sequences were aligned using CLUSTALW2 and viewed using GeneDock software. Forward and reverse primers are shown as black arrows with primer names mentioned above. Symbol (//) represents discontinuity in multiple sequence alignment. Highly conserved amino acid blocks are shaded in black.
Table 1.
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.
Figure 3.
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.
(A). Amplified overlapping PCR amplicons using A.terreus 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). Restriction digestion pattern of ligated overlapping PCR fragments in TA vector with EcoRI 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. (C). Pair-wise sequence alignment of primer walking DNA sequence with the un-reviewed AOx sequence information from A.terreus 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. (D). 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 EcoRI and HindIII restriction sites, respectively.
Figure 4.
Nucleotide and deduced amino acid sequence of AOx from A.terreus MTCC6324.
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.
Figure 5.
Purification profile of apo-rAOx and western blot analysis.
(A). Purification profile of apo-rAOx using Nickel affinity chromatography. Lane M is protein molecular weight marker, lane L1 is crude solubilized supernatant, lane L2 is the unbound fraction from column, lane L3 is the washed flow through fraction, lane L4 –L9 are the elution fraction from column. Purified protein band at ∼76 kDa is shown with a black bar. (B). Western blot of the purified rAOx against N- and C-terminal 6× polyhistidine tags. Lane M is a protein molecular weight marker and lane L1 is the developed ∼76 kDa rAOx blot on PVDF membrane shown against a black bar.
Figure 6.
Fluorescence spectra and Circular dichroic spectra of refolded rAOx with co-factor FAD as holoenzyme.
(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.
Figure 7.
DLS analysis of rAOx, cAOx from Pichia pastoris and BSA for 0 h and 24 h incubation, respectively at 16°C.
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. (A). 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 A1), confirms the onset of aggregation. Panel A2 shows the 0 h DLS signal of cAOx containing high aggregation reflected by a broad peak at diameter (d) = ∼1000 nm. Panel A3 represents the aggregation profile of BSA at 0 h. (B). Panel B1, shows the highly aggregated complex formed after 24 h incubation of rAOx with major peak shift to diameter (d) = ∼1000 nm. Panel B2 shows the constant aggregation profile of cAOx with no major peak shift when compared to 0 h data. Panel B3 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.
Figure 8.
Studies on temperature, pH optima, thermal and pH stability of rAOx.
Effect of temperature (A) and pH (B) on the activity of rAOx was studied using 5 mM ρ-anisyl alcohol in 100 mM sodium phosphate buffer. Data are the mean of three (n = 3) independent experiments. (C) Thermal stability of rAOx when incubated for 1 h at different termperature. Residual activity (%) was calculated taking enzyme activity value (in U mg−1) with 5 mM ρ-anisyl alcohol as substrate in 100 mM sodium phosphate buffer, pH 6.0 as 100%. (D) pH stability of rAOx at different pH buffer monitored for 1 h. Residual activity was calculated as mentioned above.
Figure 9.
I-TASSER predicted ab-initio model of apo-rAOx docked with its co-factor FAD.
(A). 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) 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 (C). 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.
Figure 10.
3D superimposition of predicted rAOx holoenzyme model with that of the holoenzyme aryl alcohol oxidase crystal structure (PDB id: 3FIM).
The 3D superimposition of our predicted FAD bound rAOx model (shown in yellow color ribbon structure) with the chain B of crystal structure of holoenzyme AOx (shown in green color ribbon structure) from P. eryngii (PDB id: 3FIM) using Molsoft ICM browser (www.molsoft.com). Both the FAD molecules from respective protein models and the ρ-methoxybenzyl alcohol as the docked substrate molecule to the model rAOx from A.terreus MTCC6324 are shown as Corey-Pauling-Koltun (CPK) model with its individual binding pockets highlighted in white scaffolds.
Figure 11.
Docking view of modeled rAOx (FAD docked) with its alcohol substrates.
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 (A), (B), (C) and (D) shows the close-up docking view generated by Molegro Virtual Docker version 4.0.2 (CLC bio-Qiagen company) of ρ-methoxybenzyl alcohol; m-methoxybenzyl alcohol; 3,4 dimethoxybenzyl alcohol and benzyl alcohol, respectively.
Table 2.
Steady-state kinetic parameters of in-vitro refolded recombinant alcohol oxidase from E.coli.