Sphingobacterium sp. T2 Manganese Superoxide Dismutase Catalyzes the Oxidative Demethylation of Polymeric Lignin via Generation of Hydroxyl Radical

Article abstract of DOI:10.1021/acschembio.8b00557

Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. ¹⁸O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-β-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative ³¹P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to ³¹P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation.

Full text of DOI:10.1021/acschembio.8b00557

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Article
Sphingobacterium sp. T2 manganese superoxide dismutase catalyses the
oxidative demethylation of polymeric lignin via generation of hydroxyl radical
Goran M. M. Rashid, Xiaoyang Zhang, Rachael C. Wilkinson, Vilmos
Fülöp, Betty Cottyn, Stéphanie Baumberger, and Timothy D.H. Bugg
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00557 • Publication Date (Web): 24 Sep 2018
Downloaded from http://pubs.acs.org on September 26, 2018
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Sphingobacterium sp. T2 manganese superoxide dismutase catalyses the oxidative
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demethylation of polymeric lignin via generation of hydroxyl radical
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Goran M. M. Rashid1#, Xiaoyang Zhang1#, Rachael C. Wilkinson2, Vilmos Fülöp2, Betty
Cottyn3, Stéphanie Baumberger3, and Timothy D.H. Bugg1*
1Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.
2School of Life Sciences, University of Warwick, Coventry CV4 7AL, U.K.
3Institut JeanꢀPierre Bourgin, INRA, AgroParisTech, CNRS, Université ParisꢀSaclay, 78000,
Versailles, France.
*Corresponding Author: Prof. Timothy D.H. Bugg, Department of Chemistry, University of
Warwick, Coventry CV4 7AL, U.K.
Email T.D.Bugg@warwick.ac.uk; Tel 44ꢀ2476 573018
#These authors contributed equally to the work described in the paper.
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Abstract:
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Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes
which exhibit unprecedented activity for lignin oxidation, but via an unknown mechanism.
Enzymatic treatment of lignin model compounds gave products whose structures were indicative of
arylꢀCα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol
oxidation. 18O labelling studies on the SpMnSODꢀcatalysed oxidation of lignin model compound
guiaiacylglycerolꢀβꢀguaiacyl ether indicated that the oxygen atom inserted by the enzyme is derived
from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P
NMR spectroscopy demonstrated 20ꢀ40% increases in phenolic and aliphatic OH content,
consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for
hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the
release of approximately 1 molar equivalent of hydroxyl radical by SpMnSOD1. Four amino acid
replacements in SpMnSOD1 were investigated, and A31H or Y27H siteꢀdirected mutant enzymes
were found to show no lignin demethylation activity according to 31P NMR analysis. Structure
determination of the A31H and Y27H mutant enzymes reveals the repositioning of an Nꢀterminal
protein loop, leading to widening of a solvent channel at the dimer interface, which would provide
increased solvent access to the Mn centre for hydroxyl radical generation.
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Keywords: lignin depolymerisation; manganese superoxide dismutase; Sphingobacterium sp. T2;
hydroxyl radical; demethylation
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Introduction
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The aromatic heteropolymer lignin is a major constituent (15ꢀ25%) of plant cell wall
lignocellulose, and represents a possible raw material for generation of renewable aromatic
chemicals by depolymersation. Although a number of chemical depoymerisation methods have
been proposed,1 few biochemical methods are available for lignin depolymerisation. Extracellular
lignin peroxidase and manganese peroxidase enzymes and multiꢀcopper dependent laccase enzymes
from whiteꢀrot basiodiomycete fungi are known to oxidise lignin and lignin model compounds2, but
more recently, bacterial enzymes for lignin degradation have emerged.3 Bacterial Dypꢀtype
peroxidase enzymes have been identified in Rhodococcus jostii RHA1,4 Amycolatopsis sp. 75iv25
and Pseudomonas fluorescens Pfꢀ56 that can oxidise polymeric lignin, and multiꢀcopper oxidases
with activity for lignin oxidation have been reported from Streptomyces coelicolor7 and
Ochrobactrum sp..8
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Rashid et al. have previously identified two extracellular manganese superoxide dismutase
enzymes from ligninꢀdegrading bacterium Sphingobacterium sp. T29 which show activity for
oxidation of organosolv and Kraft lignin, and lignin model compounds10. A number of aromatic
products were identified, arising from CαꢀCβ and arylꢀCα oxidative bond cleavage, and a
mechanism was proposed involving generation of hydroxyl radical via oneꢀelectron reduction of
hydrogen peroxide10. The conventional catalytic mechanism for manganese superoxide dismutase
involves reduction of one equivalent of superoxide to peroxide by Mn(II), and oxidation of a second
equivalent of superoxide to dioxygen by Mn(III).11,12 E. coli manganese superoxide dismutase is
known not to further reduce peroxide to hydroxyl radical, although the copper/zinc superoxide
dismutase can form hydroxyl radical.13 Nevertheless, precedent for the attack of hydroxyl radical
upon lignin can be found in brown rot fungi, which use Fenton chemistry to produce hydroxyl
radical14,15. The crystal structure of SpMnSOD1 was determined, but the active site of this enzyme
was essentially superimposable upon that of E. coli MnSOD, which does not oxidise lignin10.
In this paper we report a more detailed study of the mechanism of oxidation of polymeric lignin
and lignin model compounds, and the identification of specific amino acid replacements in
SpMnSOD1 that are required for the unusual reactivity of this enzyme.
Results
Transformation of lignin model compounds
Recombinant SpMnSOD1 was incubated with several dimeric and monomeric lignin model
compounds, in the presence of potassium superoxide in DMSO. As reported previously10,
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transformation of βꢀaryl ether lignin dimer 1 by SpMnSOD1 was found to generate
methoxyhydroquinone (2, GCꢀMS retention time 19.3 min) via oxidative arylꢀCα bond cleavage,
vanillic acid (3, GCꢀMS retention time 22.7 min) via oxidative CαꢀCβ bond cleavage, and guaiacol
(4, GCꢀMS retention time 13.8 min), as shown in Figure 1. Transformation of biphenyl model
compound bivanillin (5) by SpMnSOD1 was found to generate 5ꢀcarboxyvanillic acid (6, GCꢀMS
retention time 27.4 min) via oxidative ring cleavage, and 5ꢀhydroxyvanillic acid (7, GCꢀMS
retention time 27.3 min), and protocatechuic acid (8, GCꢀMS retention time 15.8 min).
Transformation of β−β lignin dimer pinoresinol (9) by SpMnSOD1 was found to generate vanillic
acid (3, GCꢀMS retention time 22.7 min) via oxidative CαꢀCβ cleavage, protocatechuic acid (8,
GCꢀMS retention time 15.8 min), and 2ꢀhydroxyethylguaiacylketone (10, GCꢀMS retention time
35.8 min).
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Figure 1. Transformation of lignin model compounds by SpMnSOD1
Since protocatechuic acid (8) was detected in two of the above transformations, its possible
formation from demethylation of vanillic acid was studied via transformation of vanillic acid (3) by
SpMnSOD1. Protocatechuic acid was detected, consistent with demethylation activity. 5ꢀ
Hydroxyvanillic acid (7) was also detected, indicative of phenolic hydroxylation, and guaiacol (4),
formed by decarboxylation (see Figure 2). 3,4ꢀDimethoxybenzyl alcohol (11) was also incubated
with SpMnSOD1, generating the corresponding aldehyde (12) and acid (13) products via sidechain
oxidation, and methoxyquinone (14).
In order to probe the mechanism of oxidation of pinoresinol (9), monocyclic ketone
acetovanillone (15) was also incubated with SpMnSOD1, generating vanillic acid (3), indicating
that SpMnSOD1 can catalyse Cα−Cβ oxidative cleavage of an unactivated CꢀC bond.
Protocatechuic acid (8) was also detected, consistent with demethylation of vanillic acid.
Finally, ferulic acid (16) was also incubated with SpMnSOD1. A new peak at retention time
29.7 min was observed, with m/z 229, consistent with the formation of 2,3ꢀ
dihydroxyphenylpropanoic acid (17) via dihydroxylation of the alkene functional group of ferulic
acid. Vanillin (18) and oxalic acid (19) were also detected, whose formation could be rationalised
via oxidative CꢀC cleavage of acid 17.
Figure 2. Transformation of aromatic substrates by SpMnSOD1
In the case of βꢀaryl ether 1, the reaction mechanism was probed further via 18O labelling.
Since SpMnSOD1 is also active using hydrogen peroxide as substrate, 18Oꢀlabelled hydrogen
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peroxide was generated in situ by reaction of 18O2 with glucose oxidase and 1 mM glucose.
Transformation of lignin model compound 1 by SpMnSOD1 under an atmosphere of 18O2 in the
presence of glucose oxidase and glucose was found to generate 18Oꢀlabelled methoxyhydroquinone
2 (retention time 19.2 min, M+ 284.6 (disilylated)), with 40.3% 18O incorporation, and 18Oꢀlabelled
vanillic acid 3 (retention time 22.7 min, M+ 312.6 (disilylated)), with 39.3% 18O incorporation,
consistent with the incorporation of one atom of 18O from hydrogen peroxide into 2 and 3. The GCꢀ
MS spectra for 18Oꢀlabelled and unlabelled products are shown in Supporting Information Figure
S2.
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Evidence for release of hydroxyl radical during SpMnSOD1 catalysis
Based on the chemical structures of low molecular weight products formed from breakdown of
polymeric lignin by SpMnSOD1, Rashid et al. previously hypothesised that the lignin oxidation
activity of SpMnSOD1 could be caused by the overꢀreduction of hydrogen peroxide by Mn(II) to
hydroxyl radical10, a very powerful oxidant that is generated in brownꢀrot fungi via Fenton
chemistry in order to attack lignin14,15. In order to seek further evidence for hydroxyl radical release,
hydroxyphenylfluorescein (HPF) was used as a fluorescenceꢀbased assay for hydroxyl radical16.
Treatment of βꢀaryl ether model compound with SpMnSOD1 and hydrogen peroxide gave rise to
increased fluorescence, compared with controls lacking either enzyme or hydrogen peroxide, as
shown in Figure 3. Fluorescence signal was dependent upon protein concentration and hydrogen
peroxide concentration (see Supporting Information Figure S20), but was nonꢀlinear vs time,
maximum fluorescence being formed in most assays after 1ꢀ2 minutes. Addition of 5 mM sodium
thiosulfate, a quencher for hydroxyl radical,17 to the assay caused a decrease in observed rate (see
Supporting Information Figure S21), and the production of protocatechuic acid from vanillic acid
by SpMnSOD1 was inhibited by 70% by addition of 5 mM sodium thiosulfate, and inhibited
completely by addition of 3 µM hydroxyphenylfluorescein. Calibration of the fluorescence change
was carried out using the Fenton reaction using variable concentrations of iron (II) ammonium
sulphate with hydrogen peroxide (see Supporting Information Figure S20C),18 indicating a release
of 47 µM hydroxyl radical by 11.4 µM SpMnSOD1, a stoichiometry of 4.1 mole hydroxyl radical
per mole enzyme.
Figure 3 Hydroxyphenylfluorescein assay for hydroxyl radical detection
Reaction co-product and stoichiometry
The coꢀproduct of the enzymeꢀcatalysed reaction was also investigated. Although
demethylase enzymes usually generate formaldehyde via oxidation of the methyl group CꢀH bond,
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hydroxyl radical has been shown to react with methoxyꢀsubstituted aromatic compounds via an
ipso-reaction, with loss of methanol via CꢀO bond cleavage.19 Attempts to detect formaldehyde
using the colorimetric assay of Li et al20 gave no observed signal from 1,4ꢀdimethoxybenzene, and
only 2ꢀ3 µM formaldehyde from 1 mM vanillic acid (see Supporting Information Figure S22). In
contrast, 107 µM methanol was detected via oxidation of the methanol produced to formaldehyde,
using Pichia pastoris alcohol oxidase,21 followed by acetoacetanilide assay (Supporting Information
Figure S22).
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Reaction of 1 mM vanillic acid and 4 mM hydrogen peroxide with 11 µM SpMnSOD1 was
analysed. Estimation of hydrogen peroxide concentration using a coupled assay with P. fluorescens
peroxidase Dyp1B6 and 2,4ꢀdichlorophenol revealed that 2 mM hydrogen peroxide was consumed,
hence that after dismutation, 1 mM hydroxyl radical could be formed. HPLC analysis revealed that
110 µM vanillic acid was consumed in the reaction, and that 28 µM protocatechuic acid was formed
as the major product, with several minor products formed, including 2ꢀmethoxyhydroquinone,
guaiacol and catechol. Hence we estimate that, of the hydroxyl radical generated by the enzyme,
approximately 90% decomposes, and 10% reacts with the vanillic acid substrate to form close to
one equivalent of methanol and a range of products, of which protocatechuic acid formed by
demethylation is the major product.
Transformation of polymeric lignin by SpMnSOD1
Samples of wheat straw organosolv lignin were treated with SpMnSOD1 in the presence of
either KO2/DMSO, or H2O2 in aqueous buffer, either of which led to the visible solubilisation of the
lignin samples over 1ꢀ4 hr (see Supporting Information Figure S23), and samples were analysed by
gel permeation chromatography. Surprisingly, no significant change in apparent Mw was measured
(see Table 1): in both cases the observed Mw had changed by <1% of the value in the absence of
oxidant after 1ꢀ4 hr. Therefore, although some monomeric products are formed,10 the increased
solubility of the lignin polymer could not be explained by a decrease in molar mass of the polymer.
Therefore, a more detailed structural investigation was undertaken on Protobind alkali lignin treated
with SpMnSOD1 and H2O2 in aqueous buffer.
Table 1. GPC analysis of lignin treated by SpMnSOD1
In order to assess the proportion of lignin units involved only in βꢀaryl ether bonds,
thioacidolysis was applied to the lignin samples before and after SpMnSOD1 treatment. The S/G
ration was found to be 1.14 in both cases, indicating that no major structural modification of the
lignin polymeric backbone had taken place, which was consistent with the apparent absence of
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depolymerisation observed by GPC. This result suggested that the changes in solubility might be
caused by oxidative demethylation of methoxy groups in lignin units to hydroxyl groups. The
hydroxyl content of lignin can be assessed experimentally by phosphitylation of free hydroxyl
groups (see Figure 4A), followed by 31P NMR spectroscopy, in which the integration and chemical
shift of the resulting NMR signals estimates the content of hydroxyl groups, and its classification
into aliphatic OH, phenolic OH (S type, G type, H type) and carboxyl OH22. After treatment with
SpMnSOD1 and H2O2, analysis by 31P NMR spectroscopy revealed 20ꢀ40% increases in aliphatic
OH and phenolic OH (see Figure 4B), and a slight increase in carboxyl OH content, consistent with
oxidative demethylation (phenolic G, S OH). The increase in total OH content was supported by
FTꢀIR analysis of treated vs untreated lignin samples, which also showed increases of the 3300ꢀ
3400 cmꢀ1 band (see Supporting Information Figure S24).
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In contrast, recombinant Pseudomonas putida manganese superoxide dismutase SodA, which did
not exhibit lignin modification, showed either no change or 5ꢀ10% decreases in OH content. Thus,
the increased solubility of the lignin after SpMnSOD1 treatment was assigned to increased OH
content due to oxidative demethylation.
Figure 4. Changes in lignin OH content after treatment by SpMnSOD1
Identification of amino acid residues in SpMnSOD1 responsible for demethylation activity
Although the crystal structure of SpMnSOD1 revealed that the active site in the vicinity of
the Mn(II) cofactor was essentially superimposable upon that of E. coli MnSOD,10 an amino acid
sequence alignment of SpMnSOD1 (see Table 2) showed the presence of some amino acid
replacements close to Mn(II)ꢀbinding ligands, which are located in the vicinity of the active site
(see Figure 5). Adjacent to Mn(II) ligand Hisꢀ26, a conserved Hisꢀ27 residue is replaced by Tyr,
and conserved Hisꢀ31 that is situated close in space, on the next turn of an αꢀhelical structure, is
replaced by Ala in SpMnSOD1. A third replacement close in space to Tyrꢀ27 in SpMnSOD1 is the
sidechain of a Gln residue found as Leuꢀ4 in EcMnSOD1. A fourth replacement adjacent to Mn(II)
ligand Hisꢀ76 is the replacement of Ser or Ala in bacterial MnSOD sequences by Gluꢀ77 residue in
SpMnSOD1. These four residues were therefore selected for siteꢀdirected mutagenesis, and
SpMnSOD1 mutants Q4L, Y27H, A31H and E77S were generated, in which each of these four
residues was changed to the corresponding residue found in E. coli MnSOD1.
Table 2. Partial amino acid sequence alignment showing amino acid replacements in SpMnSOD1.
Figure 5. Location of amino acid replacements found in Sphingobacterium sp. MnSOD1.
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Each of the mutant enzymes was expressed and purified, and the mutant enzymes showed
65ꢀ110% of the wildꢀtype superoxide dismutase activity (see Supporting Information Figure S25).
Samples of Protobind lignin were treated with each SpMnSOD1 mutant and H2O2, and samples
were analysed by 31P NMR spectroscopy as described above. As shown in Figure 4C, treatment by
mutants E77S and Q4L gave virtually identical increases in OH content to the wildꢀtype
SpMnSOD1 enzyme, however, treatment with mutants A31H or Y27H gave no increases in OH
content, in fact gave slightly lower OH content than untreated lignin. There is therefore a clear
difference in behaviour between the mutants, indicating that both Alaꢀ31 and Tyrꢀ27 are essential
for demethylation activity. The 20ꢀ30% decrease in OH content in these mutants might be due to
some repolymerisation of the lignin, and we note that 5ꢀ10% decrease in OH content was observed
for P. putida SodA, compared with untreated samples. Assay of mutants A31H and Y27H with the
hydroxyphenylfluorescein assay in the presence of hydrogen peroxide showed no significant
reaction above background (see Figure 3), indicating that these mutants do not generate hydroxyl
radical, and no production of protocatechuic acid from vanillic acid was detected for either mutant
enzyme via HPLC analysis.
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The Y27H (PDB code: 6GSB) and A31H (PDB code: 6GSC) SpMnSOD mutant enzymes were
then crystallised, and their structures determined by Xꢀray crystallography to a resolution of 1.37
and 1.32 Å respectively. Mutants show a very high level of structural identity with the wild type
with a global RMSD of 0.20 (over 206 CA atoms) and 0.20 (over 205 CA atoms) for Y27H and
A31H respectively (Figure 6).
Figure 6. Superposition of SpMnSOD1 wild type structure with Y27H (A) and A31H (B).
Superposition of the structures of A31H and Y27H SpMnSOD1 mutants on the structure of
wildꢀtype SpMnSOD1 highlighted a shift between the Nꢀterminal loop and the parallel ⍺ꢀhelix.23
There are two points where the distance between the Nꢀterminal loop and parallel ⍺ꢀhelix are most
notably altered in the mutants compared to wild type (Figure 7). The first shift is only observed in
the Y27H mutant where there is a widening between residue H/Y27 on the ⍺ꢀhelix and residues
P6/L7 on the Nꢀterminal loop (Figure 6B) amounting to a 0.7 Å increase in the distance compared
to the wild type (Table 3). At the second position there is a narrowing between residue A/H31 on
the ⍺ꢀhelix and residue Q4 on the Nꢀterminal loop. This narrowing is more substantial (~2 Å
decrease compared to wild type) than the difference observed at the first position and interestingly
occurs in both the Y27H and A31H mutant.
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Figure 7. Superposition of SpMnSOD1 wild type (light blue) structure , A31H (green) and Y27H
(orange) mutants.
Table 3. Distances in Ångström between the Nꢀterminal loop and parallel ⍺ꢀhelix measured
between the carbon backbones at the two mutation points.
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This alteration in the interaction between the Nꢀterminal loop and the parallel ⍺ꢀhelix does not
significantly alter the positioning of the Mn coꢀordination or ‘gateway’ residues.24,25 Solvent
channels in the wild type and mutants were modelled using Betacavityweb.26 This analysis showed
a major solvent channel between the two monomers in the wild type and mutants. The solvent
channel situated in the middle of the homodimer has previously been shown to be important in the
structure and function of SOD enzymes.24,25 A modest narrowing for the bottleneck of this solvent
channel was observed for the mutants compared to the wild type (Table 4). Further analysis of
channels originating from the pocket between the dimers were calculated using CAVER
(Supplementary Figure S26).26 This analysis showed the number of calculated channels decreased
in the mutants compared to wild type, with 8 predicted channels in the wild type compared to 7 for
both the A31H and Y27H mutant. These observations might suggest a decrease in solvent
accessibility for the mutants compared to the wild type. We hypothesise that the observed change in
the interaction between the Nꢀterminal loop and the parallel ⍺ꢀhelix within these mutants causes a
slight alteration in the orientation of this ⍺ꢀhelix resulting in a small shift in several residues,
including those involved in the solvent channel.
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Table 4. Calculated bottlenecks for the major solvent channel in SpMnSOD1 wild type and mutants
by Betacavityweb.26
Discussion
The involvement of Sphingobacterium sp. T2 manganese superoxide dismutase enzymes in
lignin oxidation10 is unusual, since manganese superoxide dismutase is normally involved in
protection against oxidative stress, hence it was of particular interest to elucidate the catalytic
mechanism for this reaction. In this paper we provide evidence that treatment of polymeric lignin
with SpMnSOD1 causes increases in phenolic OH content, consistent with oxidative demethylation,
a reaction observed with vanillic acid (3) and observed previously with guaiacol.10 We have
elsewhere observed by FTꢀIR spectroscopy increases in OꢀH stretch of polymeric lignin treated
with the host bacterium Sphingobacterium sp. T2,28 that are consistent with demethylation activity.
Increases in aliphatic OH content were also observed by 31P NMR spectroscopy, which would not
be explained by oxidative demethylation, suggesting that some interꢀunit bond cleavage reactions
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are also taking place. The observed reaction of pinoresinol 8 with SpMnSOD1 releases alcohol 10,
hence oxidative cleavage of β−β linkages found in polymeric lignin might explain the observed
increases in aliphatic OH content. These structural changes do not affect the average molar mass of
the lignin, but would have the effect of solubilising the polymeric lignin, as we previously
observed.10 Solubilisation of lignin content in lignocellulose in vivo would allow attack by soil
bacteria on the cellulose and hemicellulose polysaccharides of lignocellulose, and could be an
valuable biotechnological method for lignin solubilisation.
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SpMnSOD1 is also shown to catalyse a range of oxidative reaction types on lignin model
compounds, including arylꢀCα and CαꢀCβ oxidative cleavage, aromatic ring cleavage, phenolic
hydroxylation, alcohol sidechain oxidation, and dihydroxylation of an alkene sidechain, consistent
with the generation of a highly reactive oxidant. Moreover, the arylꢀCα and CαꢀCβ oxidative
cleavage reactions of lignin model compound 1 were shown to involve 18O incorporation from
H218O2, and we have provided evidence for the release of 1 molar equivalent of hydroxyl radical by
SpMnSOD1, providing a molecular mechanism for this reactivity. Hydroxyl radical is known to
cause phenol hydroxylation,29 demethylation reactions,30 and addition reactions of alkenes,31 so the
observed reactions are each consistent with the generation of hydroxyl radical as a reactive oxidant
by SpMnSOD1. We therefore propose that hydroxyl radical is generated in SpMnSOD1 by oneꢀ
electron reduction of hydrogen peroxide, as shown in Figure 8, which then acts as a diffusible
oxidant to demethylate lignin, and we have shown that the byꢀproduct of this reaction is methanol,
formed via an ipsoꢀsubstitution by hydroxyl radical. Generation of hydroxyl radical from hydrogen
peroxide has also been reported previously in copperꢀzinc superoxide dismutase, although not in E.
coli manganese superoxide dismutase.13 Possible mechanisms involving hydroxyl radical for
oxidation of DDVA and pinoresinol by SpMnSOD1 are shown in Supporting Information (Figures
S28, S29).
The molecular basis for this remarkable change in function of this enzyme is shown to involve
two point mutations in SpMnSOD1: replacement of Hisꢀ31 found in E. coli MnSOD to Ala, and
replacement of Hisꢀ27 to Tyr. The structures of A31H and Y27H mutants of SpMnSOD1 show
changes in structure in an Nꢀterminal protein loop near the active site, causing a change in angle of
helix 1 at the dimer interface. There are known to be extensive interactions at the dimer interface of
manganese superoxide dismutase that are important for catalysis,24,25 including Tyrꢀ34 close to the
residues implicated here, which is the proton donor for catalytic turnover.32 Widening of the solvent
channel at the dimer interface would facilitate solvent access to the Mn(II) centre (see Supporting
Information Figure S24), which would be needed in order to deliver protons for hydroxyl radical
formation. It is likely that these two mutations would also change the charge balance at the active
site, which would alter the redox potential of the Mn(II) centre. Alignment of SpMnSOD sequences
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from a range of bacteria (see Supporting Information Figure S30) indicates that these two mutations
are not found in MnSOD sequences from bacterial ligninꢀdegrading strains such as Rhodococcus
jostii RHA1 and Pseudomonas putida KT2440, indicating that those organisms probably do not use
this strategy for lignin oxidation. The H27Y mutation is observed in some other Gramꢀnegative
bacteria such as Pseudomonas fluorescens, Comamonas testosteroni and Burkholderia xenovorans,
but replacement of both residues is only observed within the Bacteroides phylum, with B.
thetaiotaomicron containing H27Y and H31L mutations.
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Sphingobacterium sp. T2 also produces a second extracellular manganese superoxide enzyme,
which also has lignin oxidation activity.10 The sequence of this enzyme does not contain either of
the point mutations noted above, however, it does contain a deletion of 46 amino acids at the N
terminus, relative to SpMnSOD1, and 23 amino acids relative to E. coli MnSOD (see Table 2). The
Proꢀ6 and Leuꢀ7 residues that are altered in position in SpMnSOD1 are therefore completely absent
in SpMnSOD2, hence this Nꢀterminal deletion would completely remove the Nꢀterminal loop, and
we hypothesise that this would increase solvent access to the Mn centre via a different structural
change. Therefore it appears that there are two different solutions to this change in activity that are
present in the same organism. The biological production of hydroxyl radical in SpMnSOD1 is a
remarkable mechanism, but illustrates the powerful oxidative chemistry needed to attack the
chemically inert lignin structure, such as the Fenton chemistry used in brownꢀrot fungi, and the
intriguing solutions that Nature has found to achieve this feat.
Experimental
Enzyme purification
Gene encoding SpMnSOD1 enzyme has been cloned without signal peptide using forward 5'ꢀ
CACCCAATTTAAACAGACCCꢀ3' and Reverse 5'ꢀTTATTTTTTCAAGGCTTTCTCATATCG ꢀ3'
primers and transformed into E. coli BL21 strain. The enzyme was overexpressed (using 1 mM
IPTG) from recombinant plasmid (pET151 containing mature SpMnSOD1). The Hisꢀtagged protein
purified to near homogeneity by using Immobilised ion affinity chromatography (IMAC) under
native conditions (NiꢀNTA column, Qiagen), as described previously.10
Site-directed mutagenesis of SpMnSOD1
Siteꢀdirected mutagenesis was carried out using the QuikChange II XL SiteꢀDirected Mutagenesis
Kit (Agilent), following the manufucturer’s instructions, using PfuUltra HF DNA polymerase.
Primers used for SpSOD1 siteꢀdirected mutagenesis were as follows:
Q4L forward 5'ꢀgacgacatttgcacaatttaaactgaccccacttccatatgꢀ3'
Reverse 5'ꢀcatatggaagtggggtcagtttaaattgtgcaaatgtcgtcꢀ3'
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Y27H forward 5'ꢀgaccatggagatccaccacagcaagcatgctgcꢀ3'
Reverse 5'ꢀgcagcatgcttgctgtggtggatctccatggtcꢀ3'
A31H forward 5'ꢀatggagatccactacagcaagcatcatgcaggatatacggꢀ3'
Reverse 5'ꢀccgtatatcctgcatgatgcttgctgtagtggatctccatꢀ3'
E77S forward 5'ꢀcgggaggccactacaaccacagcctgttttggtctatcctaacꢀ3'
reverse 5'ꢀgttaggatagaccaaaacaggctgtggttgtagtggcctcccgꢀ3'
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Transformation of lignin model compounds
Lignin dimer model compounds (1.6 mM final concentration) and monocyclic aromatic
compounds (16 mM final concentration) were incubated with SpMnSOD1 (final concentration 8ꢀ10
µM) in 2.5 mL of 50 mM NH4HCO3 buffer pH 7.8 containing 1.6 mM EDTA, to which 1 mL of
saturated KO2/DMSO under N2 was added. After 1 hr, 1 mL of 1 M HCl was added to stop the
reaction.
Samples for LC/MS analysis (0.5 mL) were extracted with ethyl acetate (1 mL) and then
evaporated under reduced pressure. The organic residues were redissolved in 1:1 MeOH/H2O, and
injected onto reverse phase column Phenomenex Luna 5 ꢁm C18 (100 Å, 50 mm, 4.6 mm) on an
Agilent 1200 and Bruker HCT Ultra mass spectrometer, at a flow rate of 0.5 mL/min, monitoring at
310 and 270 nm. The solvent system was water (A) and MeOH (B) containing 1% formic acid (for
positive ionisation mode), starting with 5% of buffer B, then the following gradient: 0ꢀ30% B over
0ꢀ20 min; 30% B for 10 min; 30ꢀ100% B from 30ꢀ45 min; 100% B for 8 min; 100ꢀ5% B for 8 min.
Samples for GC/MS analysis (0.5 mL) were extracted with ethyl acetate (1 mL) and then
evaporated under reduced pressure. The residues were reconstituted with dry chloroform (1 mL),
dried (MgSO4), and then either analysed directly by GCꢀMS or silylated with N,Oꢀ
bis(trimethylsilyl)acetamide. Silylation reaction for GCꢀMS was carried out by mixing 200 µL of
samples in dry solvent with 100 µL silylation mixture of chlorotrimethylsilane (TMCS), N,Oꢀ
bis(trimethylsilyl)acetamide (BSA) (1:20) and 200 µL of pyridine at 60 oC for 1 hr and diluted 10ꢀ
fold. The analysis was performed using a gas chromatographꢀmass spectrometer (GC/MS/MS,
Varian 4000) on a Varian Factor Four column (length= 30 m, i.d= 0.25 mm, thickness= 0.25 µm).
Electron impact mass spectra (EIꢀMS) were recorded at ionization energy of 70 eV. The
temperature gradient was as follows: 50°C for 1 min; 50ꢀ300°C at the rate of 7.5°C per minute; then
maintained at 300 oC for 5.67 minutes.
18O labelling experiment
In a 20 mL glass vessel containing 4 mL phosphate buffer (50 mM, pH 7), lignin model compound
guaiacylglycerolꢀβꢀguaiacyl ether (1 mM final concentration) was incubated with SpMnSOD1 (10ꢀ
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20 µM), and glucose oxidase from Aspergillus niger (50ꢀ100 U/mL). The vessel was degassed with
nitrogen, and then saturated with 18O2 gas (Cambridge Isotope Laboratories) then glucose solution
(1 mM final concentration, degassed under N2) was added. After 1 hr the products were extracted
with EtOAc and analysed by GCꢀMS.
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GPC analysis
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Organosolv lignin (1.66 mg) was added to 1 mL 50 mM phosphate buffer (pH 7.8, with 0.1 mM
EDTA), then 2.6 uL of 1M hydrogen peroxide was added, followed by 0.4 mg purified SpMnSOD1.
The reaction incubated at 30 oC for 4 hr. After reaction, 250 µL 1M HCl was added to each sample
to terminate the enzymatic reaction. Samples were centrifuged (12,000 rpm, microcentrifuge) for 10
min, and then subjected to GPC analysis in DMF. A control incubation contained enzyme preꢀ
mixed with 250 µL 1M HCl.
Quantitative 31P NMR and sample preparation.
To prepare samples for 31P NMR experiment, 100mg Protobind alkali lignin (Green Value Ltd)
was dissolved in 40ml 50mM phosphate buffer pH 7.8 containing 0.1 mM EDTA. Then 2.0 mg of
purified SpMnSOD1 was added, and 2.6 µL 1M hydrogen peroxide was added to initiate the
reaction. The reactions were incubated at 30 oC for 2 hr. 1.6 ml 1M HCl was added to terminate the
reaction and precipitate the enzyme and lignin. The samples were then freezeꢀdried. A control
reaction containing no enzyme was also carried out, reactions were carried out in duplicate.
Derivatization of the lignin samples with 2ꢀchloroꢀ4,4’,5,5’ꢀtetramethylꢀ1,3,2ꢀdioxaphospholane
(TMDP, SigmaꢀAldrich, France) was performed according to Granata et al..15 Lignin samples
(approximately 20 mg) were dissolved in 400 µL of a mixture of anhydrous pyridine and deuterated
chloroform (1.6:1 v/v). Then 150 µL of a solution containing cyclohexanol (4.05 mg/mL) and
chromium(III) acetylacetonate (3.90 mg/mL) was added, which served as internal standard and
relaxation reagent respectively and 75 µL of TMDP. NMR spectra were acquired on a Bruker
Biospin Avance III 400 MHz spectrometer. A total of 128 scans were acquired with a delay time of
6 s between successive pulses. The spectra were processed using Topspin 3.1. All chemical shifts
are reported relative to the product of TMDP with cyclohexanol, which has been observed to give a
doublet at 145 ppm referenced from the waterꢀTMDP signal (132.2 ppm). The content of hydroxyl
groups (in mmol.gꢀ1) was calculated on the basis of hydroxyl groups contained in the internal
reference cyclohexanol and by integration of the following spectral regions: aliphatic hydroxyls
(151–146 ppm), syringyl phenolic hydroxyls (144ꢀ141 ppm), guaiacyl phenolic hydroxyls (140.5–
138.5), pꢀhydroxyphenyl phenolic hydroxyls (138.4–137.0 ppm), and carboxylic acids (136.6ꢀ133.6
ppm).
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HPF (Hydroxyphenyl fluorescein) assay for hydroxyl radical detection
Purified SpMnSOD1 (24ꢀ145 µg) was added to a solution of betaꢀaryl ether lignin model
compound (100 µM) in 250 µL 50mM phosphate buffer pH 8.0 containing 0.1 mM EDTA, and 3
µM HPF (Hydroxyphenyl fluorescein, SigmaꢀAldrich). Then 10 µl of 100 mM hydrogen peroxide
was added to generate signal. 200 µl of the reaction mixture was monitored in a Hidex 96ꢀwell
microtitre plate reader (excitation wavelength: 485 nm; emission wavelength: 535 nm) and
monitored at 30 s intervals for 10 min. Calibration was carried out using 5ꢀ40 µM ammonium iron
(II) sulphate with 3 µM HPF (hydroxyphenyl fluorescein) in 250 µl 50 mM phosphate buffer pH
7.5. Then 2.5 µl 100 mM hydrogen peroxide was added to generate hydroxyl radical, and the
reaction monitored as above.
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Detection of co-products of SpMnSOD reaction
Formaldehyde as a possible product was analysed from vanillic acid and 1,4ꢀdimethoxy
benzene (1 mM final concentration) as substrates with purified SpMnSOD1 (100 µg) in 50 mM
phosphate buffer pH 8.0 at 30 °C, with acetoacetanilide in the presence of ammonia using the
method of Li et al.21 Samples (20 µL) were added to microtiter plates containing ethanolic
acetoacetanilide (80 µL of 100 mM solution), to which 100 µL of ammonium acetate (4 M) were
added. The signals were monitored monitored using Hidex microtiter plate reader (excitation
wavelength: 355/40 nm; emission wavelength: 460/20 nm) at 30 sec intervals for 10 min. A series
of standard solutions (10, 20, 50 and 100 µM) of formaldehyde (SigmaꢀAldrich) were used for
calibration.
Methanol as a possible product was analysed by the method of Arrett,22 via conversion to
formaldehyde using alcohol oxidase from Pichia pastoris, SigmaꢀAldrich) then formaldehyde was
determined as described above. Samples (20 µL) initially incubated with alcohol oxidase (5 µL of
100 U) in 100 µL of 50 mM phosphate buffer pH 7.5 at 30 °C, then 20 µL was taken for
formaldehyde determination as above. A series of standard solutions of methanol (20, 40, 60, 80
and 100 µM) were used for calibration.
The amount of remaining hydrogen peroxide from the reaction of SpMnSOD1 with vanillic
acid was determined using 2,4ꢀdichlorophenol (2,4ꢀDCP) and peroxidase enzyme (Dyp 1B from
Pseudomonas fluorescens).6 Samples (20 µL) were added to microtiter plates wells containing 5
µL of 2,4ꢀDCP (100 mM), 5 µL of 4ꢀaminoantipyrine (35 mM) and 2 µL of Dyp 1B in 200 µL
potassium acetate (50 mM) buffer pH 5.5. The absorbance was monitored using Hidex microtiter
plate reader at 510 nm for 10 min. A series of solutions of hydrogen peroxide (5, 10, 15, 30, 60 and
120 µM) were used for calibration.
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Structure determination of Y27H and A31H mutant enzymes
Pure recombinant Y27H SpMnSOD1 mutant (20 mg/mL) and A31H SpMnSOD1 mutant (17
mg/mL) in 20 mM Tris pH 8 was subjected to manual crystallisation screening using 24 well
screens designed around crystallisation conditions found for the wild type.10 1 ꢀL of protein was
mixed with 1 ꢀL of crystallisation solution. Plates were incubated at 18 °C and small rodꢀshaped
crystals appeared after 3ꢀ4 months. Y27H crystals grew in 160 mM ammonium citrate dibasic, 26%
PEG 3350 and A31H crystals grew in 130 mM ammonium citrate, 26% PEG 3350. Crystals were
removed from drops using a mounted Litholoop (Molecular Dimensions), cryoprotected in
crystallisation solution containing 10% ethylene glycol/glycerol for Y27H and 15% ethylene glycol
for A31H and flashꢀfrozen in liquid nitrogen.
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Xꢀray diffraction data to a resolution of 1.37 Å for Y27H and 1.32 Å for A31H were collected at
100K at the beamline I03 at the Diamond Light Source, U.K. using a Pilatus 6M detector. All data
were indexed, integrated and scaled using the XDS package.33 Further data handling was carried out
using the CCP4 software package.34 Refinement of the structure was carried out by alternate cycles
of manual refitting using Coot35 and Refmac,36 using the initial model of the isomorphous wildꢀtype
structure (PDB code: 5a9g).10 Water molecules were added to the atomic model automatically using
ARP,37 at the positions of large positive peaks in the difference electron density, only at places
where the resulting water molecule fell into an appropriate hydrogen bonding environment.
Restrained isotropic temperature factor refinements were carried out for each individual atom. The
polypeptide chain was traced through electron density maps (2Fo - Fc and Fo – Fc), for residues ꢀ4
to 201 for mutant Y27H, and residues ꢀ2 to 202 for mutant A31H. Data collection and refinement
statistics are given in Table 4.
Supporting Information is available, containing GCꢀMS and LCꢀMS spectra of SpMnSOD1
reaction products, supporting biochemical assay data, GPC and FTꢀIR data, and supporting
crystallographic data (30 figures, 22 pages). This material is available free of charge via the internet
at http://pubs.acs.org.
Acknowledgements. This work was supported by BBSRC research grants BB/M025772/1 and
BB/M003523/1. This project has received funding from the BioꢀBased Industries Joint Undertaking
under the European Union Horizon 2020 research and innovation programme under grant
agreement 720303, and was supported in part by a PhD studentship (to G.M.M.R.) funded by the
Kurdistan Regional Government (KRG) Ministry of Higher Educarion and Scientific Research. The
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IJPB benefits from the support of the LabEx Saclay Plant SciencesꢀSPS (ANRꢀ10ꢀLABXꢀ0040ꢀ
SPS). Crystallographic data were collected at beamline I03 at Diamond Light Source, UK and we
acknowledge the support of beam line scientists K. McAuley and M. Williams.
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21. Arrett, D. I. M. B. (2004) Comparison of three colorimetric reagents in the determination of
methanol with alcohol oxidase: application to the assay of pectin methylesterase. J. Agric. Food
Chem. 52:3749−3753
22. Granata, A. and Argyropoulos, D.S. (1995). 2ꢀChloroꢀ4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ
dioxaphospholane, a reagent for the accurate determination of the uncondensed and condensed
phenolic moieties in lignin. J. Agric. Food Chem. 43:1538ꢀ1544.
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23. Maiti, R., Van Domselaar, G.H., Zhang, H., and Wishart, D.S. (2004). SuperPose: a simple
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Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase.
Biochemistry 40:15ꢀ27.
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25. Whittaker, M.M. and Whittaker, J.W. (1998). A glutamate bridge is essential for dimer stability
and metal stability and metal selectively in manganese superoxide dismutase. J. Biol. Chem.
273:22188ꢀ22193.
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26. Kim, JꢀK., Cho, Y., Lee, M., Laskowski, R.A., Ryu, S.E., Sugihara, K., and Kim D.S. (2015)
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(2012). CAVER 3.0: A Tool for Analysis of Transport Pathways in Dynamic Protein
Structures. PLOS Comput. Biol. 8:10
28. Konstantopoulou, M., Slator, P.J., Taylor, C.R., Wellington, E.M., Allison, G., Harper, A.L.,
Bancroft, I., and Bugg, T.D.H. (2017). Variation in susceptibility to microbial lignin oxidation
in a set of wheat straw cultivars: influence of genetic, seasonal and environmental factors.
Nordic Pulp & Paper Research Journal 32:493ꢀ507.
29. Grootveld, M. and Halliwell, B. (1986). Aromatic hydroxylation as a potential measure of
hydroxyl radical formation in vivo: identification of hydroxylated derivatives of salicylate in
human body fluids. Biochem. J. 237:499ꢀ504.
30. FraserꢀReid, B., Jones, J.K.N., and Perry, M.B. (1961). The demethylation of sugars with
hydrogen peroxide. Can. J. Chem. 39:555ꢀ563.
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spectra of radicals obtained by addition of amino and hydroxyl radicals to alkenes. J. Chem.
Soc. 530ꢀ533.
32. Whittaker, M.M. and Whittaker, J.W. (1997) Mutagenesis of a proton linkage pathway in
Escherichia coli manganese superoxide dismutase. Biochemistry 36:8923ꢀ8931
33. Kabsch, W. (2010) XDS. Acta Crystallographica Section D 66:125ꢀ132
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Providing programs for protein crystallography. Methods in Enzymology 277:620ꢀ633
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36. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997) Refinement of macromolecular
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4
structures by the maximumꢀlikelihood method. Acta Crystallogr. D Biol. Crystallogr. 53:240ꢀ
255
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37. Langer, G. G., Cohen, S. X., Lamzin, V. S., and Perrakis, A. (2008) Automated macromolecular
model building for Xꢀray crystallography using ARP/wARP version, Nature Protocols 3:1171ꢀ
11795.
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Table 1. Molecular weight (Mw, g molꢀ1) measurement by gel permeation chromatography of wheat
straw organosolv lignin samples treated by SpMnSOD1.
5
6
7
8
9
KO2/DMSO
4,870
H2O2
5,120
5,060
5,070
5,090
Lignin + buffer
Lignin + enzyme + buffer
+ oxidant, 1 hr
4,970
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52
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54
55
56
57
58
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60
4,910
+ oxidant, 4 hr
4,990
Table 2. Partial amino acid sequence alignment, showing amino acid replacements in SpMnSOD1
and SpMnSOD2 (green) situated close to Mn ligands Hisꢀ26, Hisꢀ76 (yellow), and active site
residues Hisꢀ30, Tyrꢀ34 and Asnꢀ75 (cyan).
4
26
76
SpMnSOD1
SpMnSOD2
SODM_THET8
SODM_STRPY
SODM_ECOLI
H6MT45_GORPV
QFKQTPLPYAYDALEGAIDAKTMEIHYSKHAAGYTANLNKAI
----------------------MEIHHDRHHQAYVDNLNKAI
PFKLPDLGYPYEALEPHIDAKTMEIHHQKHHGAYVTNLNAAL
AIILPELPYAYDALEPQFDAETMTLHHDKHHATYVANTDAAL
SYTLPSLPYAYDALEPHFDKQTMEIHHTKHHQTYVNNANAAL
EYTLPDLPYDYAALEPHISGRIMELHHDKHHATYVKGANDTL
GHYNHELFWSILTP
GHYNHKLFWSILSP
GHLNHSLFWRLLTP
GHLNHALFWELLSP
GHANHSLFWKGLKK
GHTNHSIFWKNLSP
Table 3. Distances in Ångström between the Nꢀterminal loop and parallel ⍺ꢀhelix measured
between the carbon backbones at the two mutation points.
Distance of Y/H27 to P6 (Å)
Distance of A/H31 to Q4
(Å)
10.1
8.2
7.8
7.3
SpMnSOD1 Wild Type
Y27H SpMnSOD1
A31H SpMnSOD1
E. coli SOD
9.3
10.3
9.4
8.7
Table 4. Calculated bottlenecks for the major solvent channel in SpMnSOD1 wild type and mutants
by Betacavityweb.19
Channel Bottleneck (Å)
SpMnSOD1 Wild Type
Y27H SpMnSOD1
A31H SpMnSOD1
1.432
1.413
1.409
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Table 5. Crystallography data collection and refinement statistics
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4
5
6
7
8
Y27H
A31H
Data collectiona
Space group
Cell dimensions
a, b, c (Å)
P21
P21
9
46.68, 59.09, 75.21
90, 90.47, 90
0.97948
37ꢀ1.45 (1.53ꢀ1.45)
368354 (52448)
71340 (5108)
0.065 (0.569)
13.7 (2.1)
46.53, 58.61, 75.07
90, 90.37, 90
0.97623
40ꢀ1.32 (1.39ꢀ1.32)
937258 (132118)
94757 (6973)
0.090 (0.951)
14.3 (2.0)
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60
α, β, γ (°)
Wavelength (Å)
Resolution (Å)
Observations
Unique reflections
Rsym
I/σ(I)
Completeness (%)
Redundancy
98.5 (97.1)
5.2 (5.1)
100.0 (100.0)
9.9 (9.6)
Refinement
Rcryst
Reflections used
Rfree
Reflections used
Rcryst (all data)
Nonꢀhydrogen atoms
0.149 (0.283)
68524 (4922)
0.169 (0.290)
2816 (186)
0.150 (0.243)
90988 (6698)
0.166 (0.256)
3769 (275)
0.150
0.151
3872 (including 2 Mn2+
628 waters
&
3839 (including 2 Mn2+
529 waters
&
Bꢀfactors
Protein
Water
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
DPI coordinate error (Å)
8.3
33.5
16.5
32.2
0.014
1.6
0.060
0.014
1.6
0.045
aValues in parentheses are for highestꢀresolution shell.
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Figure Legends
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Figure 1. Transformation of dimeric lignin model compounds by SpMnSOD1. GCꢀMS and LCꢀMS
data for the products identified are shown in Supporting Information (Figures S1ꢀS7).
8
9
Figure 2. Transformation of monomerc aromatic substrates by SpMnSOD1. GCꢀMS and LCꢀMS
data for the products identified are shown in Supporting Information (Figures S8ꢀS19).
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Figure 3. Hydroxyphenylfluorescein assay for hydroxyl radical detection, carried out as described
in Experimental section, using purified SpMnSOD1 or mutant A31H or Y27H enzyme (100 µg)
and betaꢀaryl ether lignin model compound (100 µM) and 4 mM hydrogen peroxide in 50mM
phosphate buffer pH 8.0. Control assays contained either no enzyme or no hydrogen peroxide.
Figure 4. Changes in lignin OH content after treatment by SpMnSOD1. A, schematic illustration of
phosphitylation method for determination of OH content by 31P NMR spectroscopy; B, OH content
of Protobind alkali lignin before and after treatment by SpMnSOD1 and P. putida SodA; C, OH
content of Protobind alkali lignin before and after treatment with wildꢀtype and siteꢀdirected mutant
SpMnSOD1 enzymes.
Figure 5. Location of amino acid replacements found in Sphingobacterium sp. MnSOD1.
Figure 6. Superposition of SpMnSOD1 wild type with mutants A31H (A) and Y27H (B).
Manganese ions are depicted as purple spheres. Residues with altered conformation between wild
type and mutant are depicted as stick models. Density (σ level 2) shown for residue 31 for A31H
and for residue 27 for Y27H, with comparable residues in the wild type depicted alongside.
Figure 7. Superposition of SpMnSOD1 wild type (light blue), A31H (green) and Y27H (orange)
mutants. Distance between Nꢀterminal loop and parallel ⍺ꢀhelix are shown in blown up image.
Figure 8. Generation of hydroxyl radical by SpMnSOD1 and demethylation of lignin units.
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OH
O
CO2H
OH
HO
7
8
OMe
OMe
OMe
OMe
9
OH
OH
OMe
OH
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OH
3
2
4
1
aryl-Cα cleavage
Cα-Cβ cleavage
OHC
OMe
HO2C
OMe
HO2C
OMe
OH
HO2C
OH
OH
OH
OH
OH
CO2H
OH
6
8
7
OHC
OMe
oxidative ring cleavage
5
demethylation
OMe
OH
O
OH
CO2H
CO2H
O
O
OMe
OH
OH
OMe
OH
OH
10
Cβ-Cβ cleavage
9
3
8
HO
Cα-Cβ cleavage
OMe
demethylation
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CO2H
CO2H
CO2H
1
2
3
4
5
OH
OMe
HO
OMe
OH
OMe
OH
OH
OH
6
7
8
9
3
8
7
phenolic
hydroxylation
4
demethylation
decarboxylation
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CH2OH
CHO
CO2H
O
OMe
OMe
OMe
OMe
OMe
OMe
OMe
O
12
13
11
14
alcohol sidechain oxidation
Cα-Cβ cleavage
O
CO2H
CO2H
OMe
OH
OMe
OH
OH
OH
15
3
8
Cα-Cβ cleavage
demethylation
CO2H
CO2H
HO
CHO
OH
CO2H
CO2H
OMe
OH
OMe
OMe
OH
17
dihydroxylation
OH
16
18
19
Cα-Cβ cleavage
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180000
160000
140000
120000
100000
80000
Wild-type SpMnSOD1
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27
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36
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42
Mutant Y27H
Mutant A31H
60000
40000
0.0 1.0 2.0 3.9 5.4 9.3 11.3 13.3 16.2 20.2 24.1 29.1
Time (min)
A31H
Y27H
SpMnSOD1
Control -Enz
Cont -H2O2
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A
B
O
P
O
O
OH
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2
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O
P
O
O
HO
MeO
O
O
O
OH
MeO
O
P
O
MeO
MeO
1.8
1.6
1.4
1.2
1
9
No enzyme
SpMnSOD1
PpSodA
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0.8
0.6
0.4
0.2
0
Aliphatic OH
Phenolic S OH
Phenolic G OH
Phenolic H OH
Carboxylic acid
1.8
No enzyme
WT enzyme
E77S mutant
Q4L mutant
A31H mutant
Y27H mutant
C
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
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Phenolic G OH
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Figure 6
81x172mm (300 x 300 DPI)
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Figure 7
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