Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases

Article abstract of DOI:10.1021/ja210657t

Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O ₂-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose.

Full text of DOI:10.1021/ja210657t

Communication
pubs.acs.org/JACS
Oxidative Cleavage of Cellulose by Fungal Copper-Dependent
Polysaccharide Monooxygenases
William T. Beeson,†,⊥ Christopher M. Phillips, Jamie H. D. Cate,†,‡,§,∥ and Michael A. Marletta*,†,‡,§,∥,#
‡,⊥
†
‡
§
Department of Chemistry, Department of Molecular and Cell Biology, and California Institute for Quantitative Biosciences,
University of California, Berkeley, California 94720, United States
∥
Division of Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
*
S Supporting Information
modification to catalysis is unknown. The other equatorial
coordination sites are occupied by the Nε of another histidine
residue and a water molecule. A buried proximal tyrosine acts as
the fifth ligand. The bidentate histidine binding motif is also
present in CBP21, a structurally related chitin-degrading
enzyme with less than 10% sequence identity to fungal
ABSTRACT: Fungal-derived, copper-dependent polysac-
charide monooxygenases (PMOs), formerly known as
GH61 proteins, have recently been shown to catalyze the
O -dependent oxidative cleavage of recalcitrant polysac-
2
charides. Different PMOs isolated from Neurospora crassa
were found to generate oxidized cellodextrins modified at
the reducing or nonreducing ends upon incubation with
cellulose and cellobiose dehydrogenase. Here we show that
the nonreducing end product formed by an N. crassa PMO
is a 4-ketoaldose. Together with isotope labeling experi-
ments, further support is provided for a mechanism
involving oxygen insertion and subsequent elimination to
break glycosidic bonds in crystalline cellulose.
15
PMOs. CBP21 was reported to use Zn or Mg to catalyze
oxygen-dependent cleavage of chitin. The products of CBP21
are aldonic acids, and it was reported that one oxygen atom
16
from molecular oxygen was incorporated into the products. In
view of the similarity to PMOs, it is likely that Cu is the
relevant metal in CBP21.
The oxidative chemistry catalyzed by fungal PMOs likely
leads to incorporation of molecular oxygen into the products.
We proposed a mechanism by which PMOs insert molecular
oxygen into C−H bonds adjacent to the glycosidic linkage,
13
ignocellulosic biomass is a large, renewable, and untapped
leading to elimination of the adjacent carbohydrate moiety.
1
L
resource for production of fuels and chemicals. Enzymatic
conversion of lignocellulose to monosaccharides is a slow and
expensive process, largely because cellulose is an insoluble
Two classes of PMOs were described. Type-1 PMOs generate
products oxidized at C1, probably initially in the lactone form,
17
which is then hydrolyzed spontaneously or enzymatically to
yield an aldonic acid. Following phosphorylation, gluconic acid
could be metabolized by the pentose phosphate pathway. Type-
2 PMOs generate products oxidized at the nonreducing end.
Formation of a 4-ketoaldose could result from oxidative
cleavage proceeding by the same general mechanism proposed
for oxidation at C1. Another report with Thermoascus
aurantiacus GH61A also observed oxidation at C1 and on the
2
crystalline substance. For cellulase-catalyzed hydrolysis to
occur, a cellulose chain must be separated from the crystalline
surface and then drawn into the active site of the enzyme,
where Asp or Glu residues function in general acid/base
catalysis. The separation of the glucan chain from crystalline
cellulose has been proposed to be the slow step in enzymatic
3
hydrolysis of cellulose.
11
Oxidative enzymes have recently been shown to be
important for enzymatic degradation of cellulose and have
been identified in multiple transcriptomic and proteomic
nonreducing end, proposed to be at C6. No data has been
reported that identifies the position on the carbohydrate of the
nonreducing-end oxidation catalyzed by type-2 PMOs. This
contribution presents evidence that further elucidates the
mechanism of PMO action through the use of isotope-labeling
experiments and definitive identification of the nonreducing-
end product of a type-2 PMO.
4
−9
analyses.
Polysaccharide monooxygenases (PMOs), previ-
ously called GH61 proteins, are a recently discovered class of
copper-dependent metalloenzymes that oxidatively cleave
glycosidic bonds on the surface of cellulose without requiring
10−14
separation of a glucan chain.
These enzymes require
To show oxygen insertion from O
containing 5.0 μM type-1 PMO from Neurospora crassa
NCU08760), 2.0 mM ascorbic acid, 50 mM ammonium
, reaction mixtures
2
molecular oxygen and reducing equivalents from cellobiose
1
0,13
(
dehydrogenase.
drive the reaction.
A chemical reductant can also be used to
PMOs have been shown to augment the
1
1,13
bicarbonate (pH 7.8), and 5.0 mg/mL phosphoric acid swollen
cellulose (PASC) were prepared anaerobically. The solutions
were sealed in 1.0 mL vials, and then the headspace was
10,12,13
hydrolysis of cellulose by cellulases,
and a deeper
mechanistic understanding of these enzymes could be used to
reduce further costs of cellulosic biofuels.
18
replaced with O on a Schlenk line or opened to atmospheric
2
oxygen. Reactions were allowed to proceed at 40 °C for 1 h and
The metal center of PMOs contains a conserved type-2
13
11
then analyzed by LC−MS as previously described. Cellulase
copper binding site. The bound copper is coordinated by a
bidentate N-terminal histidine residue through the amino
terminus and Nδ. The N-terminal histidine is also methylated
at Nε, but the contribution of this post-translational
Received: November 17, 2011
Published: December 20, 2011
©
2011 American Chemical Society
890
dx.doi.org/10.1021/ja210657t | J. Am. Chem.Soc. 2012, 134, 890−892

Journal of the American Chemical Society
Communication
and PMO reactions are typically carried out at pH 5.0. A more
basic pH was chosen here in order to limit the exchange of bulk
water into the products. Figure 1 shows mass spectra
Figure 1. Mass spectra illustrating the incorporation of 18O from 18O2
into the aldonic acid products of a type-1 PMO (NCU08760). Assays
were performed with (A) 1 O or (B) O , and the products were
6
18
Figure 2. Product identification of a type-2 PMO. (A) Schematic
showing the experimental design for identification of PMO products
by oxidation or reduction followed by TFA hydrolysis. (B) HPLC
chromatogram (Dionex PA-200) showing the products of a type-2
PMO (NCU01050) and CDH after hypoiodite oxidation and TFA
hydrolysis. (C) HPLC chromatogram (Dionex PA-20) of NCU01050
products following reduction with sodium borohydride and TFA
hydrolysis.
2
2
analyzed by LC−MS. Shown is a ladder of aldonic acid products (DP
3
−7). DP = degree of polymerization. The inset is an expanded region
around the mass of the cellotrionic acid ion.
confirming incorporation of 1 atom of 18O into the aldonic
acid products, providing evidence that the enzyme functions as
a monooxygenase. Similar experiments in which the ascorbic
acid was substituted with Myceliophthora thermophila cellobiose
dehydrogenase 2 (MtCDH-2) (Supplementary Figure 1) were
also performed. 1 O incorporation into the products was also
observed at both pH 5.0 and 7.8. A type-2 nonreducing-end
PMO (NCU01050) did not produce products labeled with
removed by centrifugation, and TFA was added to a
concentration of 2.0 M. Oxidized oligosaccharides were heated
at 121 °C for 1 h and then dried under a stream of nitrogen and
washed twice with isopropanol. Products were then dissolved in
0.1 M sodium hydroxide and analyzed by Dionex high-
performance anion-exchange chromatography (HPAEC) as
8
1
8
O; however, the predicted 4-ketoaldose or dialdose products
13
were present as hydrates in aqueous solutions and would
previously described (Figure 2b). The absence of glucuronic
acid ruled out a PMO product containing a C6 aldehyde.
The formation of a 4-ketoaldose by NCU01050 was
investigated next. Direct hydrolysis of the NCU01050 and
MtCDH-2 reaction products generated three peaks on a
Dionex PA-200 column (Supplementary Figure 2). As
expected, glucose and gluconic acid were formed from the
internal carbohydrates and oxidation of the reducing end by
CDH. The third peak, eluting at 6.4 min, was absent in control
reactions with NCU08760 and MtCDH-2 and probably is the
PMO-oxidized nonreducing-end carbohydrate. If a ketone
functional group were introduced in the ring, reduction with
sodium borohydride would convert it back to a epimeric
mixture of alcohols at that position (Figure 2a). Galactose is the
C4 epimer of glucose and can be readily separated from glucose
using a Dionex PA-20 column. Initial reaction products from
the NCU01050 reaction were treated with 10 mg/mL sodium
borohydride in 500 mM ammonium hydroxide for 2 h at room
temperature. After reduction, residual borohydride was
removed by addition of glacial acetic acid. The reduced sample
was dried under a stream of nitrogen and hydrolyzed using
TFA as described above. Analysis of the monosaccharide
products using a Dionex HPAEC confirmed the formation of
galactose (Figure 2C). In control experiments where
1
8,19
rapidly exchange with bulk water.
The identity of the nonreducing-end product generated by
the type-2 PMO (NCU01050) was then investigated. Both C6
and C4 oxidation have been proposed to be the nonreducing-
11,13
end modification catalyzed by some PMOs;
however, no
evidence beyond mass measurements has been provided to
support these hypotheses. To distinguish between these two
possible products, a chemical modification strategy was used
(
Figure 2a). It is well-established that carbohydrates containing
a C6 aldehyde can be oxidized to the corresponding uronic
acids upon treatment with mild oxidants such as Br or I .
2
0,21
If
2
2
the nonreducing-end product contained a C6 aldehyde,
oxidation with hypoiodite would convert it to a carboxylic
acid. After chemical hydrolysis with trifluoroacetic acid (TFA),
three monosaccharide products would be produced: glucuronic
acid, derived from the nonreducing end; glucose, from internal
carbohydrates; and gluconic acid, from oxidation at the
reducing end. NCU01050 (5.0 μM) was incubated with 0.2
μM MtCDH-2, 5.0 mg/mL PASC, and 50 mM sodium acetate
buffer (pH 5.0) at 40 °C for 10 h to generate nonreducing-end
products. PASC was removed by centrifugation, and the
products were then oxidized with hypoiodite as previously
2
1
described. After oxidation, residual iodine was removed by
addition of excess silver carbonate. Precipitated material was
8
91
dx.doi.org/10.1021/ja210657t | J. Am. Chem.Soc. 2012, 134, 890−892

Journal of the American Chemical Society
Communication
NCU08760 was substituted for NCU01050, 10-fold less
galactose was formed (Supplementary Figure 3).
Together, these results provide support for the reaction
pathway shown in Scheme 1. Type-1 PMOs insert oxygen at
ACKNOWLEDGMENTS
■
We thank S. Bauer and M. Pauly for helpful discussions on
carbohydrate analysis. W.T.B. and C.M.P. were recipients of
NSF predoctoral fellowships. This work was funded by a grant
from the Energy Biosciences Institute to J.H.D.C. and M.A.M.
Scheme 1. Proposed Reaction Pathway for Oxidative
Cleavage of Cellulose by PMOs
REFERENCES
■
(
1) Perlack, R. D.; Wright, L. L.; Turhollow, A. F.; Graham, R. L.;
Stokes, B. J.; Erbach, D. C. Biomass as Feedstock for a Bioenergy and
Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual
Supply; Oak Ridge National Laboratory: Oak Ridge, TN, 2005.
(2) Himmel, M. E.; Ding, S. Y.; Johnson, D. K.; Adney, W. S.;
Nimlos, M. R.; Brady, J. W.; Foust, T. D. Science 2007, 315, 804.
(
(
3) Wilson, D. B. Curr. Opin. Biotechnol. 2009, 20, 295.
4) Tian, C.; Beeson, W. T.; Iavarone, A. T.; Sun, J.; Marletta, M. A.;
Cate, J. H. D.; Glass, N. L. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
2157.
2
(5) Eastwood, D. C.; et al. Science 2011, 333, 762.
(6) MacDonald, J.; Doering, M.; Canam, T.; Gong, Y.; Guttman, D.
S.; Campbell, M. M.; Master, E. R. Appl. Environ. Microbiol. 2011, 77,
211.
7) Vanden Wymelenberg, A.; Gaskell, J.; Mozuch, M.; Sabat, G.;
Ralph, J.; Skyba, O.; Mansfield, S. D.; Blanchette, R. A.; Martinez, D.;
Grigoriev, I.; Kersten, P. J.; Cullen, D. Appl. Environ. Microbiol. 2010,
76, 3599.
3
(
C1, while the NCU01050 type-2 PMO inserts oxygen at the 4
position. After oxygen insertion, the glycosidic bond is
destabilized and likely broken by an elimination reaction,
which may be catalyzed by the PMO or occur spontaneously.
This elimination is irreversible because the carbon on the
reducing or nonreducing end has been oxidized.
(
(
8) Berka, R. M.; et al. Nat. Biotechnol. 2011, 29, 922.
9) Phillips, C. M.; Iavarone, A. T.; Marletta, M. A. J. Proteome Res.
2
(
011, 10, 4177.
10) Langston, J. A.; Shaghasi, T.; Abbate, E.; Xu, F.; Vlasenko, E.;
Sweeney, M. D. Appl. Environ. Microbiol. 2011, 77, 7007.
11) Quinlan, R. J.; et al. Proc. Natl. Acad. Sci. U.S.A. 2011, 108,
5079.
12) Harris, P. V.; Welner, D.; McFarland, K. C.; Re, E.; Navarro
Insertion of oxygen likely occurs following PMO-mediated
hydrogen abstraction from C1 or C4 to generate a substrate
radical. This substrate radical could recombine with a copper−
oxo species in the PMO active site, hydroxylating the glucan
chain to form the intermediate shown. The C−H bond
dissociation energy for C4 is higher than that for C1 and likely
similar to those for the remaining positions on the pyranose
ring, suggesting that a PMO could insert oxygen at any
(
1
(
Poulsen, J. C.; Brown, K.; Salbo, R.; Ding, H.; Vlasenko, E.; Merino, S.;
Xu, F.; Cherry, J.; Larsen, S.; Lo Leggio, L. Biochemistry 2010, 49,
3305.
2
2
(
13) Phillips, C. M.; Beeson, W. T. IV; Cate, J. H.; Marletta, M. A.
ACS. Chem. Biol. 2011, 6, 1399.
14) Karkehabadi, S.; Hansson, H.; Kim, S.; Piens, K.; Mitchinson,
C.; Sandgren, M. J. Mol. Biol. 2008, 383, 144.
15) Vaaje-Kolstad, G.; Houston, D. R.; Riemen, A. H.; Eijsink, V. G.;
van Aalten, D. M. J. Biol. Chem. 2005, 280, 11313.
16) Vaaje-Kolstad, G.; Westereng, B.; Horn, S. J.; Liu, Z.; Zhai, H.;
position. The advantage of oxygen insertion at C1 or C4 is
that a simple elimination reaction leads to bond cleavage.
Oxygen insertion at other positions would require the
involvement of additional amino acid residues on the surface
of the PMO to potentiate bond cleavage, and there would be
nothing to prevent reformation of the glycosidic bond on the
cellulose surface. There is a vast amount of sequence variation
in the PMO superfamily, and many highly cellulolytic fungi
express more than 10 different PMOs during growth on
(
(
(
Sorlie, M.; Eijsink, V. G. Science 2010, 330, 219.
(17) Beeson, W. T. IV; Iavarone, A. T.; Hausmann, C. D.; Cate, J. H.
D.; Marletta, M. A. Appl. Environ. Microbiol. 2011, 77, 650.
(18) Campbell, R. E.; Tanner, M. E. Angew. Chem., Int. Ed. Engl.
4
,8
cellulose. The residues controlling regiospecificity in PMOs
are unknown. Future work to identify the reaction products of
divergent members of the PMO superfamily may reveal what
factors control the position of oxygen insertion.
1
997, 36, 1520.
(19) Naundorf, A.; Klaffke, W. Carbohydr. Res. 1996, 285, 141.
(20) Kelleher, F. M.; Bhavanandan, V. P. J. Biol. Chem. 1986, 261,
1
1045.
21) Kelleher, F. M.; Bhavanandan, V. P. Carbohydr. Res. 1986, 155,
89.
(22) Blanksby, S. J.; Ellison, G. B. Acc. Chem. Res. 2003, 36, 255.
(
ASSOCIATED CONTENT
Supporting Information
■
*
S
Additional control experiments, experimental methods, and
complete refs 5, 8, and 11. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
marletta@scripps.edu
Present Address
#
The Scripps Research Institute, 10550 N. Torrey Pines Road,
BCC-555, La Jolla, CA 92037.
Author Contributions
⊥
These authors contributed equally.
8
92
dx.doi.org/10.1021/ja210657t | J. Am. Chem.Soc. 2012, 134, 890−892