Polycyclic ketone monooxygenase from the thermophilic fungus Thermothelomyces thermophila: A structurally distinct biocatalyst for bulky substrates

Article abstract of DOI:10.1021/jacs.6b12246

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a wellestablished tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.

Full text of DOI:10.1021/jacs.6b12246

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Communication
Polycyclic Ketone Monooxygenase from the
Thermophilic Fungus Thermothelomyces thermophila:
A Structurally Distinct Biocatalyst for Bulky Substrates
Maximilian JLJ Fürst, Simone Savino, Hanna M. Dudek, J. Ruben Gomez Castellanos,
Cora Gutiérrez de Souza, Stefano Rovida, Marco W. Fraaije, and Andrea Mattevi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b12246 • Publication Date (Web): 23 Dec 2016
Downloaded from http://pubs.acs.org on December 26, 2016
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Polycyclic Ketone Monooxygenase from the Thermophilic Fungus
Thermothelomyces thermophila: A Structurally Distinct Biocatalyst
for Bulky Substrates
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Maximilian J.L.J. Fürst,‡[a] Simone Savino, ‡[b] Hanna M. Dudek,[a] J. Rúben Gómez Castellanos,[b]
Cora Gutiérrez de Souza[c], Stefano Rovida,[b] Marco W. Fraaije,*[a] and Andrea Mattevi.*[b]
[a]Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG, Groningen, The Netherlands
[b]Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy
[c] Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands
Supporting Information Placeholder
nantly produced and characterized so far, only two enzymes
ABSTRACT: Regio- and stereoselective Baeyer-Villiger oxi-
stand out by their stability: phenylacetone monooxygenase
from Thermobifida fusca5 (PAMO) and the recently discov-
ered CHMO from Thermocrispum municipale.6 Yet, these
biocatalysts have a restricted substrate scope, being mainly
active on small aromatic or cyclic aliphatic ketones. From an
application point of view, it would be attractive to have ac-
cess to a robust BVMO acting on structurally demanding
compounds, as it is the case for many pharmaceuticals.
In an effort to exploit the genome of thermophiles for the
discovery of new enzymes with interesting catalytic proper-
ties, we considered Thermothelomyces thermophila ATCC
42464. This genome-sequenced, thermophilic fungus effi-
ciently degrades cellulose and other compounds derived
from plant biomasses and is becoming an attractive organism
for large-scale protein production.7 We identified a gene
encoding for a protein containing the typical BVMO finger-
print.8 The gene is located in a cluster of hypothetical genes
with unknown functions. A BLAST search in the UniProt
database revealed ten characterized proteins with significant
sequence identities (37-44%). Four are monooxygenases in-
volved in the biosynthesis of meroterpenoids in Aspergillus.9-
dations are difficult to achieve by classical chemical means,
particularly when large, functionalized molecules are to be
converted. Biocatalysis using flavin-containing Baeyer-
Villiger monooxygenases (BVMOs) is a well-established tool
to address these challenges, but known BVMOs have short-
comings either in stability or substrate selectivity. We char-
acterized a novel BVMO from the thermophilic fungus Ther-
mothelomyces thermophila, determined its three-dimension-
al structure, and demonstrated its use as a promising biocat-
alyst. This fungal enzyme displays excellent enantioselectivi-
ty, acts on various ketones, and is particularly active on poly-
cyclic molecules. Most notably we observed that the enzyme
can perform oxidations on both the A and D ring when con-
verting steroids. These functional properties can be linked to
unique structural features, which identify enzymes acting on
bulky substrates as a distinct subgroup of the BVMO class.
The Baeyer-Villiger oxidation is a widely-used reaction in
organic synthesis to break carbon-carbon bonds through the
insertion of a single oxygen atom adjacent to a carbonyl moi-
ety, yielding esters or lactones from ketones. Since the in-
serted oxygen is usually derived from a peroxide or peracid,
industrial scale processes raise environmental and safety
concerns.1 Baeyer-Villiger monooxygenases (BVMOs) repre-
sent an attractive biocatalytic alternative, which uses molec-
11
Three are BVMOs from Streptomyces taking part in the
processing of the antibiotic pentalenolactone.12-13 One is a
BVMO forming the carbonate functionality in the mycotoxin
cytochalasin.14 The remaining two are the biocatalytically
characterized BVMOs from Dietzia sp.15 and S. coelicolor.16
A
correlation between sequence similarity and substrate scope
has been proposed for BVMOs,17 and indeed, a feature shared
by these enzymes is the activity on bulky, polycyclic ketones.
ular oxygen as oxidant and often displays superior chemo-
,
regio-, and enantioselectivity.2 The prototype BVMO, cyclo-
hexanone monooxygenase from Acinetobacter calcoaceticus
NCIMB 9871 (CHMO) was shown to oxidize hundreds of
small aromatic, linear, and cyclic ketones with high enantio-
selectivity.3 Furthermore, one major hurdle for industrial
application of BVMOs, the requirement of stoichiometric
amounts of NADPH as reducing cofactor, has been success-
fully addressed.4 What remains problematic, however, is the
poor solvent tolerance and thermostability of most BVMOs.
Even though close to a hundred BVMOs have been recombi-
Consistently,
a
phylogenetic analysis assigns the
T. thermophila protein to a cluster containing versatile en-
zymes active on large substrates, such as cyclododecanone
monooxygenase from Rhodococcus ruber18 (CDMO) and cy-
clopentadecanone monooxygenase from Pseudomonas
sp. HI-7019 (CPDMO) (Figure S1). Collectively, these data led
us to hypothesize that the T. thermophila protein is a BVMO
that combines two attractive properties: (i) thermostability
and (ii) activity on relatively large molecules.
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An Escherichia coli codon-optimized copy of the gene was
life of approximately 24 hours when incubating the enzyme
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cloned in two translational fusion variants: a SUMO tagged
construct and a cofactor-recycling phosphite dehydrogenase
(PTDH) fusion.20 Both constructs yielded very high expres-
sion levels in E. coli and the proteins were easily obtained in
high purity and yield using standard metal affinity chroma-
tography by exploiting the N-terminal 6xHis tag (Figure
S2A). The purified yellow protein exhibited a typical flavo-
protein spectrum with absorbance peaks at 371 and 454 nm
(Figure S2B). SDS treatment revealed non-covalently bound
FAD as cofactor and the extinction coefficient ε454 of the hol-
oenzyme was estimated to be 10.2 mM-1 cm-1. Upon mixing
the enzyme with 100 µM NADPH and the BVMO model sub-
strate bicyclo[3.2.0]hept-2-en-6-one (13), rapid consumption
of NADPH was detected (Figure S3). By monitoring NADPH
consumption in the absence of substrate, we determined an
uncoupling rate of only 0.02 s-1. We also compared the cata-
lytic behavior of the two recombinant protein variants and
determined the activity of the enzyme fused to PTDH and
without any tag (obtained by cleaving SUMO). As previously
observed, the effect of the dehydrogenase fusion was very
modest and, in fact, resulted in a slightly higher activity
(Figure S3) and unchanged uncoupling rate. Since the result-
ing self-sufficient biocatalyst can be used in reactions with-
out constant addition of NADPH, as long as phosphite is
added to drive the coenzyme regeneration reaction, all sub-
sequent experiments were performed with the PTDH-fused
enzyme. The preliminary analysis was completed by deter-
mining basic enzyme characteristics for optimal catalytic
conditions. The pH profile highlighted good stability in a
range of 6.5 - 9 with a slight preference for moderately alka-
line conditions (Figure 1A).
in buffer at 35 °C, or at room temperature with 10% dioxane
(Figure 1C). These experiments indicated that the BVMO
features a considerable degree of thermostability and only
moderate sensitivity to organic solvents.
We next investigated the enzyme’s substrate scope and se-
lectivity (fully listed in Table S1). The closely related CPDMO
and CDMO are highly active on large cyclic ketones (Figure
S1). Consistently, we found conversion of 10 and 11 (Figure
S5). The fifteen membered cyclopentadecanone (8) was fully
converted and kinetic parameters were determined:
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k
cat = 0.1 s-1 and KM = 144 µM (Figure S6). Of special interest,
we observed efficient conversions of structurally demanding
compounds, including several steroids. In particular, we test-
ed stanolone (17), which has only one carbonyl group posi-
tioned at C3 of the A-ring and observed complete conversion
after 24 h (Figure S5). As a reference, we performed the con-
version with CPDMO, for which the 3-keto-4-oxa-4a-homo
isomer was reported as the only product.22 The MS spectrum
of the resulting products with the expected mass shift of
+16 Da were the same for both enzymes (Figure S5) and we
confirmed the structure by NMR (Figure S7). With pregne-
nolone (18), a steroid harboring a carbonyl group only at the
C17 sidechain, we found close to full conversion after 24 h
(Figures 2 and S5). We again observed the +16 Da mass shift
and the product was determined by NMR to be the corre-
sponding acetate (Figure S8). When the enzyme was pre-
sented with androstenedione or androstadienedione (15 and
16), steroids with keto groups in both the A and D rings, it
selectively oxidized the D-ring to yield the pharmaceutically
relevant testo(lo)lactone (Figure S5). Again, complete con-
version was observed after 24 hours.
Table 1. Substrate scope of PockeMO
Category
Structure
Linear
ketones
cyclic
ketones
Figure 1. Effects of pH, temperature and organic solvents on
the activity of T. thermophila BVMO (PockeMO). 2 µM of
PTDH-PockeMO and 0.1 mM NADPH were mixed with 13.
Substituted
cyclic
ketones
We next sought to probe the enzyme’s thermostability. We
found the activity to be highest at 50 °C, where it was four
times faster than at room temperature (Figure 1B). Using the
ThermoFAD method21, we further observed that both native
as well as PTDH-fused enzyme have an apparent melting
temperature (Tm) of 47 °C, the second highest reported for a
BVMO after PAMO. This Tm is sustained in a pH range of 7-9
and is increased a further 2 °C upon exposing the protein to
100 µM NADP+, consistent with a tight binding of this dinu-
cleotide cofactor (Figure S4). In parallel, we evaluated the
effect of organic solvents which was probed by adding
DMSO, 1,4-dioxane, ethanol, acetone or 1-propanol. The re-
sulting reductions in Tm ranged from 1 - 5 °C to 2 - 8 °C with
5% and 10% solvent, respectively (Figure S4). The enzyme
seemed to exhibit a better tolerance towards polar solvents,
with DMSO having the least and 1-propanol the highest im-
pact on Tm. Because DMSO can act as a substrate, and the
effect of the remaining solvents was comparable, we chose
dioxane as preferred cosolvent. We monitored the enzymatic
activity as a function of time and observed a satisfactory half-
Bicyclic
ketones
Steroids
To our knowledge, this is the first report of an isolated
BVMO that is able to oxidize the keto functionalities at these
different positions. Steroid monooxygenase of Rhodococcus
rhodochrous (STMO) can only convert steroidal carbonyls
positioned on the C17 sidechain and not in the rings.23
A
homolog from Cylindrocarpon radicicola in addition is able
to lactonize the D ring.24 CPDMO accepts some steroids with
the keto group on the 3 and 17 position, but is inactive on
open chain ketones.22 The T. thermophila BVMO represents a
biocatalyst combining these abilities (Table 1, red arrows)
and we therefore named it polycyclic ketone monooxygenase
(PockeMO).
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S2). Most of the residues could be modelled, apart from loop
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607-614. PockeMO exhibits the typical BVMO domain organ-
ization with an FAD-domain (residues 1-229 and 479-655), an
NADP-domain (residues 230-314 and 417-478), and a helical
domain (residues 315-416) (Figure 3). The closest structural
homologs are STMO26 and PAMO (rmsd of 1.91 and 1.75 Å for
523 and 533 equivalent Cα atoms, respectively, with 30% se-
quence identity to both). The location of the FAD and NADP
cofactors is consistent with that of other BVMOs and the
flavin shows no significant deviation from planarity (Figure
S11).
9
Figure 2. Conversion efficiencies of PockeMO, CPDMO and
CHMO (all PTDH fused) of a mix of substrates. Efficiencies
are scaled semi-quantitatively according to analysis of GC
peaks of substrates and products into: full, good or poor con-
version, or trace amounts detectable.
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We further explored the enzyme‘s substrate profile using
several substrates frequently tested with other BVMOs (Ta-
bles 1 and S1). For this purpose, we used a highly efficient
substrate screening method, based on the conversion of a
mixture of compounds and subsequent separation and analy-
sis via GC-MS. We tested a mixture of 16 linear, aromatic,
cyclic, and polycyclic ketones (Figure S5) and could immedi-
ately identify 13 conversion products. Of the unsubstituted
cyclic ketones, PockeMO converted cyclohexanone to 100%,
but the conversion of cyclooctanone was poor in comparison.
The monosubstituted cyclic ketones 9, 10 and 12 were con-
verted with yields close to 100%. Approximately half of the
disubstituted cyclic ketone 11 was converted, whereas the
unsaturated variant of this molecule (isophorone) was not
accepted. The aromatic ketone 3 was fully converted and also
the bridged, bicyclic 14 was converted with moderate yield
(Table S1, Figure 2). Notably, although being only a poorly
accepted substrate, the linear 4-octanone (1) was exclusively
converted to the abnormal product propyl pentanoate (Fig-
ure S9). These experiments elegantly demonstrated the activ-
ity on an array of substrates of varying degree of bulkiness.
At the same time, none of the compounds seemed to exhibit
inhibitory effects on the enzyme. To put this further into
perspective, we performed the conversion of a mix of 15 ke-
tones with purified CHMO, CPDMO and PockeMO. Figure 2
shows the semi-quantitative comparison of the conversion
efficiency of the three enzymes and clearly shows PockeMO’s
excellent all-round performance as well as its particular su-
periority with larger compounds.
For BVMOs, enantio- and regioselectivity has frequently
been studied by analyzing the conversion of racemic 13 into
the respective lactones. A recent extensive study on BVMO
activity in various fungi revealed diverse selectivities and
only a moderate phylogenetic correlation.25 We found that
PockeMO (4 µM enzyme, 2 hours) completely converted
10 mM racemic 13 enantiodivergently, yielding two regioiso-
meric lactones, with ee values of 100% and 97%, respectively
(Figure S10). Thus, the enzyme exhibited both high enantio-
and regioselectivity as well as high activity towards this sub-
strate. Kinetic analysis confirmed this notion as a kcat of 3.3 s-1
was measured (Km = 0.4 mM).
Figure 3. Overall structure of PockeMO (PDB entry 5MQ6).
The N-terminal extension (residues 1-73; dark blue) is
specific to this BVMO enzyme subclass (Figure S1). FAD is
yellow and NADP+ green (nicotinamide ring is disordered).
Despite this clear sequence and structural homology with
other BVMOs and the conservation of the overall fold,
PockeMO features some striking structural peculiarities.
Firstly, a 70-residues N-terminal extension forms a long α-
helix followed by an elongated stretch that wraps around the
enzyme in the vicinity of the FAD-domain (Figure 3). The
interactions between the 44 N-terminal residues and the rest
of the protein are extensive and establish 24 H-bonds, 5 salt
bridges, and many hydrophobic contacts. Notably, this elon-
gated N-terminus is a feature of all BVMOs in the phylo-
genetic group of enzymes acting on complex molecules (Fig-
ures S1 and S12A). Secondly, there is a structural element
ranging from residue 316 to 388 whose characteristic confor-
mation reshapes the active site. This region has low sequence
similarity to the canonical BVMOs (Figure S12B), in which
this portion is structurally conserved (as exemplified by
PAMO, Figure 4A). In PockeMO, this stretch forms a charac-
teristic conformation of alternating loops and α-helices. As a
result, the substrate-binding site of PockeMO is a long and
wide tunnel-like cavity, which starts from the loop 587-624
and ends at the re-face of FAD (Figures 4B-C). The flexible
loop delimited by residues 606 and 615 serves as a gate-like
mechanism, constituting a longer element when compared
to the same topological loop in PAMO (residues 495-515).
Modeling shows that bulky molecules can readily be accom-
modated in the PockeMO‘s substrate tunnel (Figure S13).
Thus, the enzyme exhibits characteristic structural features,
which are fully consistent with the notion that it belongs to a
distinct subgroup of BVMO enzymes (Figure S1).
Given these valuable stability and catalytic properties as
well as the fact that PockeMO belongs to the structurally
uncharacterized CPDMO/CDMO subfamily of BVMOs (Fig-
ure S1), it was of interest to determine the enzyme’s three-
dimensional structure. After removal of the SUMO tag, the
protein could be crystallized as a complex with FAD and
NADP+ and its structure solved at a resolution of 2.0 Å (Table
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Figure 4. Characteristic struc-
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tural features of PockeMO. (A)
Residues 316-388 (bright blue)
cover the active site and adopt a
conformation not observed in
other BVMO structures as ex-
emplified by the superimposed
equivalent residues of PAMO
(PDB 2YLT, 234-298; orange;
Figure S11). (B) PockeMO struc-
ture cut along the substrate
entrance to outline the wide
tunnel forming the active site.
(C) PAMO active site features a
narrower channel, ranging from
the solvent exposed surface to
the active site with MES bound.
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(3) Mihovilovic, M. D.; Müller, B.; Stanetty, P. Eur. J. Org. Chem.
2002, 3711.
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In summary, we identified a novel, robust and versatile en-
zyme performing Baeyer-Villiger oxidations. PockeMO com-
bines thermostability and organic solvent tolerance with a
broad substrate profile. Specifically, the enzyme accepts
bulky and complex substrates and converts them with high
efficiency, making it a promising candidate for application as
an industrial biocatalyst. In addition, the elucidated crystal
structure not only lays the basis for enzyme engineering, but
can also be regarded as a prototype of an evolutionary and
structurally distinct group of BVMOs. Careful further analy-
sis should contribute to a better understanding of the still
largely unknown mechanism by which substrate selectivity
in BVMO is tuned. The structural and sequence features of
PockeMO can also be exploited to identify new and diverse
BVMOs evolved to process relatively large substrates.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Experimental procedures, Figures S1-S13, Tables S1, S2 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Marco W. Fraaije: m.w.fraaije@rug.nl
*Andrea Mattevi: mattevi@ipvgen.unipv.it
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
The research for this work has received funding from the
European Union (EU) project ROBOX (grant agreement n°
635734) under EU’s Horizon 2020 Programme Research and
Innovation actions H2020-LEIT BIO-2014-1.
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5
ACS Paragon Plus Environment