Science - USA (2021-12-24)

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tivity relates to the mechanism of these re-
actions. Upon the catalyst-mediated forma-
tion of free radicals, these key intermediates
can diffuse out of the sphere of the catalysts
before they can impose selectivity on subse-
quent stereoselectivity-determining steps.
Some stereo-discriminative reactions have
been reported that used noncovalent interac-
tions between tailored catalysts and specially
selected radical intermediates, but these
strategies remain typically inapplicable to
common unbiased substrates (6, 7).
Zhou et al. hypothesized that executing
free-radical chemical transformations within
an active site of an enzyme could enable
controlling stereoselectivity by the intimate
interactions between the reacting interme-
diates and the substrate binding site of the
protein scaffold (see the figure). This strat-
egy mimics the excellent stereocontrol of
natural radical enzyme reactions ( 8 ). They
used cytochromes P450 that contain the
heme cofactor previously shown to medi-
ate other atom-transfer radical reactions ( 9 ).
These enzymes have been engineered to ac-
commodate several nonnatural (albeit non-
radical) reactions with high stereoselectivity
and excellent activity (2, 3, 10, 11).
Initial evaluation of a range of heme and
nonheme iron-dependent enzymes identi-
fied promising activity of previously re-
ported P411-CIS T438S ( 2 ), a variant of P450
BM3 bearing 15 mutations, which furnished
the ATRC γ-lactam product 1 with 60:40
enantiomeric ratio (e.r.). This modest stere-
oselectivity validated the initial hypothesis,
and five rounds of site-saturated mutagen-
esis targeting the residues near the active
site delivered a highly stereoselective and
active variant P450ATRCase1 bearing in total
20 mutations. P450ATRCase1 formed the target
ATRC product (R)- 1 with excellent selec-
tivity of 97:3 e.r. and high productivity of

8000 total turnovers.
Formation of only one enantiomer of a
product is often a drawback of enzymatic
synthesis. Zhou et al. also synthesized (S)- 1 ,
the mirror image of (R)- 1 , selectively in the
presence of P450ATRCase2, an independently
evolved variant of P450 BM3 bearing 23
mutations to the protein scaffold. Most no-
tably, the engineered enzymes can accom-
modate other substrates. Not only was a
range of γ-lactam products bearing differ-
ent substituents or other aromatic rings in
place of the phenyl ring formed, but also b-
and d-lactam products were produced stere-
oselectively. As a result, P450ATRCases provide
access to these important structural motifs
present in many bioactive molecules.

For starting materials with an internal

double bond bearing different substituents,

two new stereocenters are created in the re-

action. Both the initial addition of the free

radical to the double bond (that is, the C-C

bond-forming radical addition step) and

the subsequent C-Br bond-forming halogen

rebound step, are stereodetermining. Thus,

the product could be formed as four differ-

ent stereoisomers. Zhou et al. showed that

both steps can be precisely controlled within

the active site of the evolved variants of P450

BM3. Either diastereomer (that is, non–mir-

ror image stereoisomer) of the product, such

as (S,R)- 2 or (R,R)- 2 (see the figure), can be

formed with high enantio- and diastereose-

lectivity. This extent of reaction stereocon-

trol also typically inaccessible with small-

molecule catalysts ( 6 ).

These reactions proceeded efficiently in

whole cells containing these enzymes with-

out any additional manipulation or further

purification. Whole-cell catalysts facilitate

directed evolution and enable rapid evalua-

tion of hundreds of variants should the stere-

oselectivity of the reaction need to be further

improved for any substrate. The activity of

P450ATRCases in a whole cell also opens possi-

bilities for devising unnatural biosynthetic

pathways that directly combine natural and

unnatural enzymatic transformations to

yield unnatural products ( 12 ).

The repurposing of metalloenzymes to

control the reactivity of cumbersome radi-

cal intermediates reported by Zhou et al.

suggests new opportunities for controlling

other types of the atom-transfer radical ad-

dition reactions. Intermolecular transforma-

tions are arguably the most challenging but

synthetically appealing. Given the breadth of

free-radical reactions, these findings set the

stage for the development of a myriad of ste-

reoselective processes. j

REFERENCES AND NOTES

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2. Y. Yang, F. H. Arnold, Acc. Chem. Res. 54 , 1209 (2021).


3. F. Schwizer et al., Chem. Rev. 118 , 142 (2018).


4. T. K. Hyster, Synlett 31 , 248 (2020).


5. Q. Zhou, M. Chin, Y. Fu, P. Liu, Y. Yang, Science 374 , 1612
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6. M. P. Sibi, S. Manyem, J. Zimmerman, Chem. Rev. 103 ,
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7. R. S. J. Proctor, A. C. Colgan, R. J. Phipps, Nat. Chem. 12 ,
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8. C. M. Jäger, A. K. Croft, ChemBioEng Rev 5 , 143 (2018).


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11. S. N. Natoli, J. F. Hartwig, Acc. Chem. Res. 52 , 326 (2019).


12. J. Huang et al., Nat. Chem. 13 , 1186 (2021).

ACKNOWLEDGMENTS

We acknowledge the European Research Council (ERC

Starting Grant no. 804106), the French National Research

Agency (ANR IdEx, and ANR LabEx “Chemistry of Complex

Systems“), and the Frontier Research in Chemistry

Foundation.

10.1126/science.abm8321

University of Strasbourg, CNRS, ISIS UMR 7006, 8 allée
Gaspard Monge, 67000 Strasbourg, France.
Email: [email protected]

IMMUNOLOGY

Fibrin sparks

inflammation in

the oral mucosa

Fibrin deposits in the

oral mucosa trigger

neutrophil activation and

bone destruction

By T ommaso Vicanolo and Andrés Hidalgo

I

nflammation of the oral mucosa afflicts

about half a billion individuals world-

wide and is responsible for the de-

struction of the periodontium (the soft

tissue and alveolar bone around the

teeth). Periodontitis, a severe form of

this disease, has been associated with dys-

biosis of the oral microbiota ( 1 ) and with

local deposition of fibrin, a fibrous protein

involved in blood clotting. Periodontitis is

also associated with loss-of-function muta-

tions in the gene encoding plasminogen

(PLG), a protease that degrades fibrin and

prevents thrombosis. Mechanistic links

between microbial and clotting elements

and inflammation of the oral mucosa have

remained enigmatic given the traditional

view that clotting factors function inside

blood vessels. On page 1575 of this issue,

Silva et al. ( 2 ) show that deposits of fibrin

appear in the oral mucosa in a microbi-

ota-dependent manner and function as

obligatory substrates for full activation of

neutrophils—the most aggressive and tis-

sue-destructive type of leukocyte.

Fibrinogen circulates in the blood in

large amounts released by the liver, but it

is also produced by other cells, including

the mucosal epithelium ( 3 ). Because fibrin,

the cleavage product of fibrinogen, associ-

ates both with thrombosis and with inflam-

mation, controlling fibrin concentrations

is important to preserving organismal

homeostasis. The elimination of fibrin de-

posits is mediated by the protease plasmin,

itself derived from proteolytic cleavage of

its precursor protein PLG. In addition to

periodontitis, defective fibrinolysis caused

by PLG deficiency has been associated

with ocular disease (conjunctivitis) as well

Area of Cell and Developmental Biology, Centro Nacional

de Investigaciones Cardiovasculares, Madrid, Spain.

Email: [email protected]

24 DECEMBER 2021 • VOL 374 ISSUE 6575 1559