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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.
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.
Anthon,
Comparison of three colorimetric reagents in the determination of methanol with alcohol oxidase. Application to the assay of pectin methylesterase.
2004, Pubmed
Anthon,
Comparison of three colorimetric reagents in the determination of methanol with alcohol oxidase. Application to the assay of pectin methylesterase.
2004,
Pubmed
Bankar,
Glucose oxidase--an overview.
2009,
Pubmed
Bringer,
Purification and properties of alcohol oxidase from Poria contigua.
1979,
Pubmed
Bystrykh,
Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.
1991,
Pubmed
Daniel,
Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood.
2007,
Pubmed
DeRose,
Observation of a flavin semiquinone in the resting state of monoamine oxidase B by electron paramagnetic resonance and electron nuclear double resonance spectroscopy.
1996,
Pubmed
Dijkman,
Flavoprotein oxidases: classification and applications.
2013,
Pubmed
Emsley,
Coot: model-building tools for molecular graphics.
2004,
Pubmed
Federico,
Competitive inhibition of swine kidney copper amine oxidase by drugs: amiloride, clonidine, and gabexate mesylate.
1997,
Pubmed
Forneris,
ThermoFAD, a Thermofluor-adapted flavin ad hoc detection system for protein folding and ligand binding.
2009,
Pubmed
Fraaije,
Flavoenzymes: diverse catalysts with recurrent features.
2000,
Pubmed
Geissler,
Yeast methanol oxidases: an unusual type of flavoprotein.
1981,
Pubmed
Gerstenbruch,
Asymmetric synthesis of D-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol.
2012,
Pubmed
Ghanem,
Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis.
2003,
Pubmed
Kabsch,
Integration, scaling, space-group assignment and post-refinement.
2010,
Pubmed
Kabsch,
XDS.
2010,
Pubmed
Karplus,
Assessing and maximizing data quality in macromolecular crystallography.
2015,
Pubmed
Kato,
Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures.
1976,
Pubmed
Kleywegt,
Detection, delineation, measurement and display of cavities in macromolecular structures.
1994,
Pubmed
Koch,
Crystal Structure of Alcohol Oxidase from Pichia pastoris.
2016,
Pubmed
Krissinel,
Inference of macromolecular assemblies from crystalline state.
2007,
Pubmed
Lang,
Protein structural ensembles are revealed by redefining X-ray electron density noise.
2014,
Pubmed
Linke,
An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity.
2014,
Pubmed
Macheroux,
UV-visible spectroscopy as a tool to study flavoproteins.
1999,
Pubmed
Malakhov,
SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.
2004,
Pubmed
Massey,
Active-site probes of flavoproteins.
1980,
Pubmed
Mattevi,
Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity.
1997,
Pubmed
McCoy,
Phaser crystallographic software.
2007,
Pubmed
Mincey,
Presence of a flavin semiquinone in methanol oxidase.
1980,
Pubmed
Murshudov,
Refinement of macromolecular structures by the maximum-likelihood method.
1997,
Pubmed
Nguyen,
Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst.
2016,
Pubmed
Pagliaro,
From glycerol to value-added products.
2007,
Pubmed
Pettersen,
UCSF Chimera--a visualization system for exploratory research and analysis.
2004,
Pubmed
Pickl,
The substrate tolerance of alcohol oxidases.
2015,
Pubmed
Pollegioni,
Cholesterol oxidase: biotechnological applications.
2009,
Pubmed
Pollegioni,
Properties and applications of microbial D-amino acid oxidases: current state and perspectives.
2008,
Pubmed
Romero,
Alcohol oxidation by flavoenzymes.
2014,
Pubmed
Turner,
Enantioselective oxidation of C-O and C-N bonds using oxidases.
2011,
Pubmed
Vonck,
Structure of Alcohol Oxidase from Pichia pastoris by Cryo-Electron Microscopy.
2016,
Pubmed
Waterham,
Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris.
1997,
Pubmed
Winn,
Overview of the CCP4 suite and current developments.
2011,
Pubmed
Zhou,
Chemoselective catalytic conversion of glycerol as a biorenewable source to valuable commodity chemicals.
2008,
Pubmed
van Berkel,
Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.
1994,
Pubmed
van der Klei,
Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review.
1991,
Pubmed