Combinatorial alanine substitution enables rapid optimization of cytochrome P450BM3 for selective hydroxylation of large substrates

Article abstract of DOI:10.1002/cbic.201000565

Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450_BM3 variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates.

Full text of DOI:10.1002/cbic.201000565

DOI: 10.1002/cbic.201000565
Combinatorial Alanine Substitution Enables Rapid Optimization of
Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates
Jared C. Lewis,[d] Simone M. Mantovani,[e] Yu Fu,[b] Christopher D. Snow,[a] Russell S. Komor,[a]
Chi-Huey Wong,[b, c] and Frances H. Arnold*[a]
Selective hydroxylation of CÀH bonds in organic compounds
provides an efficient means to access valuable drug metabo-
lites, natural product derivatives, and other fine chemicals.[1]
While chemical methods to accomplish this transformation
have improved, these generally require the presence of direct-
ing groups or electronic properties inherent to certain sub-
strate classes for the desired transformations to occur at all or
with useful regioselectivity.[2] Enzymes are capable of avoiding
this limitation by employing potent H-abstraction mechanisms
with selectivity imposed by specific substrate binding.[3] Fur-
thermore, systematic optimization of these catalysts by direct-
ed evolution has been demonstrated extensively and consti-
tutes a powerful advantage of these systems over small mole-
cule catalysts.[4]
(NADPH) for hydroxylation activity. These properties have led
our groups and others to expand the substrate scope of this
enzyme with the goal of creating efficient enzyme and whole-
cell catalysts for a variety of oxidative transformations.[7]
The development of BM3 variants that catalyze regioselec-
tive demethylation or demethoxymethylation of methylated or
methoxymethylated monosaccharides was recently reported
by our groups.[8] While a chemoenzymatic method employing
these enzymes provided a convenient means to access other-
wise difficult-to-synthesize monosaccharide derivatives, it also
highlighted some limitations of existing BM3 variants. For
example, while MOM-protected pentoses were compatible
with these enzymes, MOM-protected hexoses were deprotect-
ed to only a minor extent (Scheme 1). This same limitation was
Members of the cytochrome P450 monooxygenase super-
family are remarkable examples of such catalysts.[5] These en-
zymes utilize a cysteine-bound heme cofactor to catalyze a
wide range of oxidative transformations including hydroxyl-
ation and epoxidation. Cytochrome P450BM3 (BM3) from Bacillus
megaterium possesses a number of features that make it par-
ticularly attractive for applications in chemical synthesis.[6] For
example, the heme domain in which hydroxylation occurs and
the diflavin reductase domains (FMN and FAD) that contribute
electrons for oxygen activation in the heme domain are fused
in a single polypeptide chain; this improves the rate and op-
erational simplicity of BM3-catalyzed reactions. Indeed, BM3
catalyzes the subterminal hydroxylation of C12–C18 fatty acids,
its natural substrates, at rates of thousands of turnovers per
minute, making it one of the most active hydroxylases known.
BM3 is soluble, readily over-expressed in a variety of heterolo-
gous hosts, and requires only atmospheric oxygen and a
supply of nicotinamide adenine dinucleotide phosphate
Scheme 1. Structures of compounds utilized in enzyme library design and
screening: methoxymethyl (MOM)-protected xylose, hexoses, and thioglyco-
sides (1–7); alkaloids thebaine and dextromethorphan (8 and 9); trimethyl
estriol (10).
[a] Dr. C. D. Snow, R. S. Komor, Prof. F. H. Arnold
Division of Chemistry and Chemical Engineering
California Institute of Technology, Pasadena, CA 91125 (USA)
Fax: (+1)626-568-8743
encountered with a number of additional bulky compounds
including various opiate alkaloids and steroid derivatives. At-
tempts to identify enzymes compatible with these substrates
from random mutant libraries generated by error prone PCR
were complicated by the low activity of all BM3 variants exam-
ined. We hypothesized that the shape and volume of these
substrates exceeded the capacity of the enzyme active site,
and that sufficient expansion of the active site to obtain signifi-
cant improvements in activity might require more extensive
mutation than can be readily accomplished with error prone
PCR.[9]
E-mail: arnold@cheme.caltech.edu
[b] Y. Fu, Prof. C.-H. Wong
Department of Chemistry, The Scripps Research Institute
La Jolla, CA, 92037 (USA)
[c] Prof. C.-H. Wong
Genomics Research Center, Academia Sinica
Nankang, Taipei 115 (Taiwan)
[d] Dr. J. C. Lewis
Current address: Department of Chemistry
University of Chicago, Chicago, IL 60637 (USA)
An alternative approach involving extensive replacement of
bulky active site residues with alanine was, therefore, explored
as a means to obtain enzymes with activity against the afore-
mentioned compounds. This involved first selecting a thermo-
stable parent capable of tolerating extensive mutagenesis,[10]
[e] S. M. Mantovani
Current Address: Institute of Chemistry, University of Campinas
P.O. Box 6154, 13083-970 Campinas SP (Brazil)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000565.
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and identifying active site residues likely to clash with large
substrates based on computational models of these com-
pounds docked in the enzyme active site. Because it is difficult
to predict which of the chosen residues might actually impact
catalysis,[11] a combinatorial library design that includes all per-
mutations of alanine substitutions was utilized. Library mem-
bers were then screened for activity with a panel of bulky sub-
strates that possess methylated heteroatoms by detecting
formaldehyde released during P450-catalyzed removal of Me
or MOM groups.[8] These substrates thus serve as efficient
probes for enzyme activity and for compatibility of valuable
natural product scaffolds with BM3 variants.
substrate scope of the library. Each of these is relatively large,
possesses several methylated heteroatoms, and belongs to a
privileged class of compounds (i.e., monosaccharides, alkaloids,
and steroids), for which novel hydroxylation catalysts could be
highly valuable (vide infra). High-throughput screening was
carried out in 96-well microtiter plate format[8] by addition of
NADPH to solutions of the appropriate substrate and cell
lysate. Following incubation at room temperature, the reac-
tions were quenched with a basic solution of Purpald, which
reacts with the formaldehyde produced from P450-catalyzed
heteroatom demethylation to generate a purple dye. The
parent enzyme, 9-10AF87VTS, displayed only weak activity
with these substrates, and provided a signal of 20% over back-
ground in the best cases (data not shown). However, many
variants with high activity against several substrates were im-
mediately evident based on visual inspection, and the formal-
dehyde concentration (and thus the extent of demethylation)
was quantified by using a plate reader (A550nm).
BM3 variant 9-10AF87VTS, a thermostable form of an
enzyme (9-10AF87V) previously found to have activity against
a-1,2,3,4-tetramethoxymethyl xylose (1),[8] was selected as a
parent for library creation. Structures for the most stable con-
formers of 1 and previously unreactive monosaccharide sub-
strates, including 1,2,3,4,6-pentamethoxymethyl glucose (2),
galactose (3), and mannose (4), were generated by using the
program Omega.[12] The resulting conformers were then placed
into the active site of a model of 9-10AF87VTS such that their
terminal methyl CÀH bonds
Variants with 4.1–7.9-fold improvement in demethoxymethy-
lation activity toward each of the MOM-protected hexose
derivatives were identified (Table 1). Furthermore, GC or HPLC
were oriented according to the
Table 1. Sequence–activity relationship for alanine substitution.
transition state geometry for cy-
tochrome P450-catalyzed H-ab-
straction proposed by Rydberg
et al.[13] The transition state en-
semble was expanded by vary-
ing rotational degrees of free-
dom and reduced by eliminating
substrate poses that clashed
with the enzyme (within 2.5 ꢁ of
a backbone or b carbon). Inspec-
tion of the final ensemble of
prospective transition state con-
formations led to the identifica-
tion of eight residues for re-
placement with alanine: K69,
L75, M177, L181, T260, I263,
T268, and L437. Combinatorial
substitution was accomplished
Substrate
Variant
Alanine substitution (+) at residue
Fold
69
75
177
181
260
263
268
437
improvement[a]
2A1
4H9
8C7
–
–
–
+
–
+
–
–
–
+
+
+
–
+
–
–
–
–
–
–
–
+
+
–
4.4
4.1
7.9
thioglycosides (5–7)
4H5
4H9
7A1
8C7
8F11
–
–
–
–
–
+
–
+
+
–
+
–
+
–
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
+
–
–
+
2.7
3.9
2.8
2.7
3.3
+
+
–
alkaloids (8, 9)
steroid (10)
–
–
8F11
–
–
–
–
–
–
–
+
n.a.[b]
[a] Ratio of A550 measurement for reaction of each variant to that of the parent (9-10AF87VTS). Ratio for sub-
strate with maximum improvement shown. [b] Improvement identified by visual inspection; substrate insolubil-
ity complicated plate reader measurement.
by cloning fragments of the parent gene containing these resi-
dues by using degenerate primers or primer mixtures encod-
ing either the original residue or alanine at each site (see the
Supporting Information). These fragments were assembled to
generate a 28 (256) member library containing the desired ala-
nine substitutions. E. coli were transformed with the library
mixture and 767 single colonies (ca. 3ꢂ theoretical library size)
were picked and used to inoculate media in 96-well deep-well
plates. Following protein expression and cell lysis, CO binding
analysis revealed that 65% of the enzymes were properly
folded.[14] Sequences from 1% of the library indicated unbiased
incorporation of alanine at all of the desired sites, and an aver-
age alanine substitution per sequence of 3.9.
analysis of the crude mixtures indicated that these reactions
proceeded in moderate to high conversion while still maintain-
ing high regioselectivity (vide infra). On the other hand, few
variants with improved activity against the smaller pentose
substrate, 1, were obtained from this library; this is consistent
with the expanded active site providing an advantage only for
the reaction of larger substrates (see the Supporting Informa-
tion). This library also contained variants with marked improve-
ments in activity with alkaloid and steroid substrates 8, 9, and
10, despite the fact that such structures were not used in the
library design. For most substrates, the best variants possessed
at least two alanine substitutions, and in general, positions 75,
177, 181, 260, 263, and 437 proved advantageous, while ala-
nine substitution at 69, 263, and 268 did not.
Several compounds, including MOM-protected thioglyco-
sides 5, 6, and 7, thebaine (8), dextromethorphan (9),[15] and
trimethyl estriol (10; Scheme 1), were selected to probe the
Given that these variants possessed sufficient activity with
substrates 2–10 to enable their detection with the aforemen-
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tioned high-throughput screen, further enzyme optimization
was possible by using directed evolution.[4] Steroid hydroxyl-
ation is a particularly valuable reaction due to the biological
activity of these compounds and their common occurrence as
metabolites.[16] Indeed, native P450s have been used for many
years in the preparation of metabolites for both analytical
studies and pharmaceutical manufacture.[17] Because only a
single variant, 8F11, exhibited activity with the steroid deriva-
tive trimethyl estriol, a library of variants of this enzyme was
generated by using error prone PCR and screened for im-
proved demethylation of trimethyl estriol (see the Supporting
Information). This led to the identification of a variant with
four mutations, none of which was located in the enzyme
active site, with 1.6-fold improvement in activity over the 8F11.
This enzyme, F1, was found to have moderate activity with
additional steroids, including 11-a-hydroxyprogesterone (vide
infra) and testosterone acetate (data not shown). This result in-
dicated that heteroatom-methylated substrates could be used
as convenient probes for BM3 activity on related compounds
lacking these handles for high-throughput analysis.
ed in 75 and 70% yield, respectively (Table 2, entries 1 and 2).
BM3-catalyzed deprotection of such substrates significantly ex-
pands the utility of our previously reported chemoenzymatic
monosaccharide elaboration procedure[8] due to the mild con-
ditions required for chemical deprotection of MOM groups and
the potential use of the thiophenyl substituent as a leaving
group in subsequent glycosylation reactions. Demethylation
and N-substitution of opiate alkaloids is commonly used to
vary the properties of these compounds,[18] and BM3 catalysis
provides a mild and operationally simple method to accom-
plish the required demethylation step (Table 2, entries 3 and
4). Finally, selective hydroxylation of multifunctional molecules
represents a great challenge in synthetic chemistry.[1] While
P450-catalyzed hydroxylation of steroids has been demonstrat-
ed, most of these reactions require the use of whole-cell bio-
catalysts[16] or multicomponent enzyme systems.[19] On the
other hand, BM3 variant F1 enabled regio- and diastereoselec-
tive steroid hydroxylation by using a single enzyme and thus
provided a convenient platform from which additional cata-
lysts for this valuable transformation can be developed
(Table 2, entry 5).
To demonstrate the synthesis utility of the enzymes ob-
tained from combinatorial alanine substitution and error prone
PCR, preparative scale bioconversions were conducted
(Table 2). Reaction conditions previously developed in our lab-
oratory were utilized;[8] yields and selectivities of the reactions
translated well to the preparative scale. For example, site-selec-
tive deprotection of MOM-protected hexoses 6 and 7 proceed-
Together, these results demonstrate the utility of combinato-
rial alanine substitution for generation of BM3 variants with ac-
tivity against bulky, synthetically useful substrates. We have
demonstrated that the resulting enzymes have novel activities
that can be further optimized by directed evolution. Monosac-
charides, alkaloids, and steroids were all viable substrates de-
Table 2. Substrate scope and reaction selectivity.
Substrate
BM3 variant
Product
Conv. [%][a]
Select. [%][b]
Yield [%][c]
1
2
2A1
80
90
75
8C7
8C7
93
88
80
72
70
60
3
4
5
4H5
54
28
98
82
50
20
F1
[a] Conversion of starting material determined by HPLC or GC analysis of crude reaction mixture. [b] Percentage of desired product relative to additional
products determined by HPLC or GC analysis of crude reaction extracts. [c] Isolated yield of the pure product.
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[5] Cytochrome P450: Structure, Mechanism, and Biochemistry (Ed.: P. R. Ortiz
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to limit the scope of their reactivity.
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Experimental Section
For details see the Supporting Information.
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[10] J. D. Bloom, S. T. Labthavikul, C. R. Otey, F. H. Arnold, Proc. Natl. Acad.
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Acknowledgements
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J.C.L. is supported by a U.S. National Institutes of Health Path-
ways to Independence Award (1K99M087551-01A1). S.M.M. is
supported by the Fundażo Coordenadoria de AperfeiÅoamento
de Pessoal de Nꢀvel Superior (CAPES; 1756-09-5). This work was
supported by the U.S. National Institutes of Health (2R01
M068664-05A1), the U.S. Department of Energy, Office of Basic
Science, grant DE-FG02-06ER15762, and King Abdullah University
of Science and Technology (KAUST), Award No. KUS-F1-028-03.
Keywords: alkaloids
·
biocatalysis
·
cytochromes
·
monosaccharides · steroids
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Received: September 21, 2010
Published online on November 24, 2010
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