Nguyen QT et al. (2018), Structure-Based Engineering of Phanerochaete ch...

ECB-ART-46638

Biochemistry 2018 Oct 30;5743:6209-6218. doi: 10.1021/acs.biochem.8b00918.

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Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol.

Nguyen QT , Romero E , Dijkman WP , de Vasconcellos SP , Binda C , Mattevi A , Fraaije MW .

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Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active toward methanol, ethanol, and 1-propanol ( kcat = 18, 19, and 11 s-1, respectively), but showed very limited activity toward glycerol ( kobs = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at a resolution of 2.6 Å. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity toward glycerol. Among them, the F101S variant had a high kcat value of 3 s-1, retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.

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Genes referenced: LOC105446919 LOC594261 sumo2

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	Figure 1. UV-vis spectral properties of PcAOX. (A) Absorption spectra of His-SUMO-PcAOX in 50 mM Tris-HCl pH 7.0 before (solid line) and after addition of 1% (w/v) SDS (broken line). (B) Spectra for F101S PcAOX. Comparison of the absorption spectra observed after two purification processes. Different ratios of the semiquinone (SQ) and quinone forms of the flavin are observed.
	Figure 2. Effect of pH on methanol oxidation catalyzed by wild-type PcAOX. The reactions contained 40 mM Britton–Robinson buffer, 0.1 μM PcAOX, and 25 mM methanol. Activity was monitored, by following dioxygen consumption for 5 min at 23 °C, using a Hansatech Oxygraph instrument.
	Figure 3. Crystal structure of PcAOX. The PcAOX octamer is shown as surface (left) and ribbon (right) representations with each individual monomer depicted in different color. For the sake of clarity, the FAD cofactor (in yellow ball-and-stick) is shown only for the monomers in the front. (A) Top view along the 4-fold axis. (B) Side view along the 2-fold axis (this orientation is obtained by rotating the oligomer 90°, with respect to the orientation in panel (A)).
	Figure 4. Superposition of PcAOX monomer A (in deep sky blue) with that of the homologous PpAOX1 (in light gray, 52% sequence identity, PDB ID: 5HSA). The overall topology of the two structures and the active site are highly conserved. Besides a longer loop of 12 amino acids at the C-terminus in PpAOX1 (which is not visible in the figure due to the orientation), the major differences between the two proteins lie in the length of some loops that are labeled with the corresponding residues. FAD is drawn as sticks with C atoms in yellow (for PcAOX) or in light gray (for PpAOX1), O atoms in red, N atoms in blue, and P atoms in orange.
	Figure 5. Quality of the electron density. Maps were calculated after molecular replacement and 8-fold averaging and are contoured at 1.2σ level. (A) The 2.6 Å resolution map for Phe101 and the flavin in the wild-type structure. (B) The 2.5 Å resolution map for the same protein region in the structure of the F101S mutant (see Table 1). For clarity, the labels for Gln102 and Thr559 are omitted.
	Figure 6. Active site. The three panels show the active-site cavities of (A) wild-type PcAOX, (B) PpAOX1, and (C) PcAOX F101S mutant. The structures are in the same orientation. Flavin carbons are shown in yellow, protein carbons are shown in gray, oxygens are shown in red, and nitrogens are shown in blue. The shape of the cavities is depicted as a pink semitransparent surface.
	Figure 7. Determination of aldehyde production in the reactions of F101S AOX with glycerol using the Purpald assay. All spectra showed here correspond to a 5-fold dilution of the initial reaction or standard solutions (before mixing with Purpald). When additional dilutions were required (see the Materials and Methods section), the spectra were corrected to obtain those presented here. 205 mM glycerol was incubated in the presence (13 μM) and the absence of F101S AOX (as represented by the red solid line and the red broken line, respectively). The inset shows that buffer (orange line) and 0.2 mM glyceric acid (blue line) exhibited very low absorbance at 540 nm, compared to that observed for 0.2 mM glyceraldehyde (green line), after incubating with Purpald (standard solutions without AOX).
	Figure 8. Activity on glycerol for the F101S PcAOX variant (A) in the presence of NaCl and (B) during incubation for prolonged time. In panel (A), enzyme (1 μM) activity on glycerol (2 M) was determined using the HRP-coupled assay in 100 mM potassium phosphate pH 7.5 with various NaCl concentrations. In panel (B), the long-term stability for the F101S PcAOX variant was probed by incubating the enzyme (32 μM) in 100 mM potassium phosphate (pH 7.5 and 25 °C). Activity on glycerol (2 M) was determined using the HRP-coupled assay at various time points.

References [+] :

Anthon, Comparison of three colorimetric reagents in the determination of methanol with alcohol oxidase. Application to the assay of pectin methylesterase. 2004, Pubmed