Alternate-site enzyme promiscuity

Article abstract of DOI:10.1002/anie.200702751

(Chemical Equation Presented) Promiscuous reactions get around: It is shown that promiscuous enzyme-catalyzed reactions (i.e., the catalysis of distinctly different chemical transformations) can also take place at sites other than where the natural reaction occurs.

Full text of DOI:10.1002/anie.200702751

Angewandte
Chemie
DOI: 10.1002/anie.200702751
Enzyme Promiscuity
Alternate-Site Enzyme Promiscuity**
Andreas Taglieber, Horst Höbenreich, J. Daniel Carballeira, RØgis J. G. Mondi›re, and
Manfred T. Reetz*
Enzyme promiscuity means, in the broadest terms, the ability
of a given enzyme to catalyze distinctly different chemical
transformations of natural or nonnatural substrates.[1]
Although it was originally thought to be a fairly rare
event,[2] research over the last few years has uncovered
many more examples. It has also become clear that catalytic
promiscuity has implications in evolutionary relationships.[1,3]
This intriguing frontier in enzymology has several important
theoretical and practical facets. For example, the question of
how proteins in nature evolve new functions such as antibiotic
resistance or the ability to degrade man-made chemicals, both
within months or years, is of fundamental significance and has
been studied by applying the methods of directed evolu-
tion.[1c,3] Moreover, the discovery of promiscuous behavior of
wild-type (WT) enzymes or mutants thereof produced by
protein engineering has the potential of expanding the
repertoire of synthetic organic methodologies.[1]
We discovered alternate-site enzyme promiscuity unex-
pectedly when screening the thermostable enzyme tHisF from
Thermotoga maritima for promiscuous behavior. It has a (b/
a)8-barrel structure and constitutes the synthase subunit of a
bienzyme complex involved in the biosynthesis of histidine.[12]
Mechanistic studies have demonstrated that ammonia is
generated in a first step by hydrolysis of glutamine (Gln), a
process catalyzed by the tHisH subunit. The ammonia then
enters tHisF at the lower rim of the barrel structure and
moves to the relatively broad top rim where the enzyme
catalyzes a cyclization reaction in an acid/base manner
(Scheme 1). Asp11 and Asp130 were shown by site-directed
mutagenesis to be essential for this catalytic step, whereas
Asp176 was reported to be important, but not essential.[12]
In all studies reported so far,[1–3] the promiscuous (secon-
dary) reaction has been linked to the binding site of the
reaction for which the enzyme is primarily known, generally
involving some or all of the original catalytically active amino
acids or metal centers. Examples are alkaline phosphatase
catalyzed hydrolysis of p-nitrophenylsulfate,[4] aminopepti-
dase-catalyzed hydrolysis of phosphoesters,[5] lipase-catalyzed
Michael additions of N-,[6] O-,[6] S-,[6] and C-nucleophiles,[7]
aldol additions,[8] oligomerization of siloxanes,[9] racemase-
catalyzed PLP-dependent aldol additions,[10] and arylmalo-
nate decarboxylase catalyzed aldol additions.[11] Many of
these studies involve protein engineering. To the best of our
knowledge, no case of enzyme promiscuity has been reported
in which the known natural catalytically active site is not
involved. Herein we report the first example of this phenom-
enon, which we call alternate-site enzyme promiscuity.
Scheme 1. Natural reaction of the bienzyme complex tHisH–tHisF.[12]
As acid/base catalysis is involved in the natural function of
tHisF, we speculated that this robust enzyme might also show
promiscuous hydrolytic behavior at the natural binding site,
specifically esterase-like activity.[13] As a model reaction we
chose the hydrolysis of p-nitrophenyl esters 1a–e [Eq. (1)].
Activity can be measured easily and accurately by the
standard photometric assay (absorption of p-nitrophenolate
at 405 nm). The rate of the background reaction is relatively
[*] A. Taglieber, H. Höbenreich, Dr. J. D. Carballeira,
Dr. R. J. G. Mondi›re, Prof. Dr. M. T. Reetz
Max-Planck-Institut für Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr (Germany)
Fax: (+49)208-306-2985
E-mail: reetz@mpi-muelheim.mpg.de
[**] A.T. and H.H. have contributed equally. We thank R. Sterner
(Universität Regensburg) for providing the plasmids for tHisF and
mutant Cys9Ala/Asp11Cys, N. Dickmann (Max-Planck-Institut für
Bioanorganische Chemie) for MALDI-TOF measurements and M.
Rusek for performing some of the fermentations. This work was
supported by the Fonds der Chemischen Industrie, the Deutsche
Forschungsgemeinschaft (SPP 1170, Re 359/13-1), and the EU
(IBAAC MCRTN-CT-2003-505020).
Supporting information for thisarticle isavailable on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 8597 –8600
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8597
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Communications
low under the reaction conditions (buffer at pH 7.4). To
obtain unambiguous results all experiments were performed
with carefully purified protein (see the Supporting
Information). To rule out small traces of possible contami-
nation with hydrolytic enzymes (e.g., esterases, lipases,
proteases), kinetic parameters were determined before and
after treatment with
a variety of inhibitors (see the
Supporting Information). As no significant differences were
observed, contamination can be excluded. Moreover, the
observed promiscuity is not due to the classical mechanism of
hydrolytic enzymes.[13]
Figure 1 shows two important results, namely, that tHisF is
indeed capable of promiscuous esterase-like catalysis
Figure 2. X-ray crystal structure of tHisF[12a,c] showing in blue the
natural catalytic amino acids Asp11, Asp130, and Asp176, as well as
Cys9.
Figure 1. Relative activity of the tHisF-catalyzed hydrolysis of p-nitro-
phenyl esters 1a–e.
position Cys9, which could be suspected of contributing to the
promiscuous esterase-like activity. By using site-specific
mutagenesis, we prepared six mutants and tested them as
potentially promiscuous catalysts in the hydrolysis of p-
nitrophenyl butyrate 1c (Table 1).[15]
(although at lower rates than esterases or lipases),[14] and
that there is notable substrate selectivity. The highest activity
is observed when the acetate 1a is subjected to hydrolysis,
whereas octanoic acid p-nitrophenyl ester (1e) hardly under-
goes any enzyme-catalyzed acceleration beyond the back-
ground reaction. Such a trend is reminiscent of the behavior
of esterases.[13]
Table 1: Kinetic parametersof the hydrolysisof
(1c).
p-nitrophenyl butyrate
tHisF or mutant
KM
[mm]
kcat
kcat/KM
vmax
We then studied the hydrolytic kinetic resolution of the
chiral ester rac-4 [Eq. (2)]. In this case, again tHisF was found
sÀ1
]
[sÀ1 mÀ1
]
[mmminÀ1
]
WT
Asp11Ala
1.9
2.2
1.9
1.5
1.4
5.4”10À4
5.5”10À4
5.0”10À4
5.6”10À4
4.9”10À4
0.28
0.25
0.26
0.39
0.34
0.0029
0.0030
0.0027
0.0030
0.0026
Cys9Ala/Asp11Ala
Cys9Ala/Asp11Cys
Cys9Ala/Asp11Cys/
Asp130Ala
Cys9Ala/Asp11Cys/
Asp176Ala
1.6
1.9
4.5”10À4
4.1”10À4
0.28
0.22
0.0024
0.0022
Cys9Ala/Asp11Cys/
Asp130Ala/Asp176Ala
to catalyze ester hydrolysis. Enantioselectivity of this pro-
miscuous reaction turned out to be substantial: the selectivity
factor amounted to E = 29 in favor of (R)-5.
Guided by the previous structural and mechanistic
information regarding the natural function of tHisF,[12] we
set out to identify the essential amino acid residues necessary
for the promiscuous hydrolytic reaction. Figure 2 displays the
known crystal structure of tHisF,[12a,c] highlighting the amino
acids that are essential or important for natural activity
(Asp11, Asp130, and Asp176; Scheme 1) in addition to
The results summarized in Table 1 are surprising because
they show that mutations at amino acid positions essential or
important for natural tHisF activity do not influence catalytic
activity in the promiscuous ester hydrolysis. The values of kcat
/
KM remain essentially constant. Thus, the catalytic machinery
of tHisF which mediates the natural cyclization reaction
(Scheme 1) is not responsible for the observed promiscuous
hydrolytic reaction. Moreover, Cys9 can also be excluded as
playing an active role in the hydrolysis reaction.
8598
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 8597 –8600
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Angewandte
Chemie
The results also indicate that the promiscuous hydrolytic
reaction may not even occur at the binding site relevant in the
natural cyclization reaction. Owing to the different steric,
electronic, and chemical nature of the newly introduced
amino acids of the mutants, one would expect to detect at
least some influence on the kinetics of the promiscuous
reaction even if other amino acids at the natural binding
pocket of tHisF are responsible for catalysis. It should be
noted that if an effect had been found, it would not necessarily
exclude an alternate site, because mutations remote from the
true binding pocket can influence activity or the enantiose-
lectivity of enzymes.[16]
To corroborate the hypothesis regarding the actual bind-
ing site in the promiscuous hydrolytic reaction, we synthe-
sized covalent bioconjugates that can be expected to occupy
considerable space in the natural binding pocket. In contrast
to the WT tHisF, the mutant Cys9Ala/Asp11Cys has an
appropriately exposed cysteine residue that can be selectively
modified with Michael acceptors. Therefore, the maleimide
derivatives 6, 7, and 8, which have different spatial properties.
were used in the preparation of the respective bioconjugates
(Scheme 2). The resulting bioconjugates were analyzed by
Figure 3. Bioconjugates made by treating mutant Cys9Ala/Asp11Cys
with Michael acceptors 6, 7, and 8. The putative structures were
generated by manual docking of the respective ligands into the tHisF
structure.
conjugates contain fairly space-filling organic residues in the
natural binding pocket, whereas the simple maleimide-
derived analogue leads to a sterically less crowded environ-
ment. As no significant effects on the catalytic profile of the
hydrolytic reaction were observed, we conclude that promis-
cuity is not likely to occur in the binding pocket used by the
enzyme in the natural cyclization reaction, which means that
alternate-site enzyme promiscuity must be operating.[17]
In conclusion, we have discovered the first example of a
promiscuous enzyme-catalyzed reaction that does not involve
any of the catalytic amino acids of the natural enzymatic
process, nor does it appear to occur in the natural binding
pocket. Further work is required to identify the actual active
center. The present results are significant because, among
other things, they raise the question of whether promiscuity as
observed for example in the degradation of man-made
chemicals evolves in nature solely by mutations at the original
active site of the enzyme, as assumed and observed so far.[1–11]
This raises a more general question, namely whether nature
utilizes, in select cases, alternate-site promiscuity to endow an
organism with an advantage for its survival and further
evolution.[18]
Scheme 2. Bioconjugation of the tHisF mutant Cys9Ala/Asp11Cys at
position Cys11.
MALDI-TOF mass spectrometry, which showed essentially
quantitative formation of the desired chemically modified
proteins. They were then tested as catalysts in the hydrolysis
of butyrate 1c. Table 2 shows that the promiscuous catalytic
Table 2: Michaelis–Menten kinetics of the hydrolysis of 1c.
Catalyst
KM
[mm]
kcat
k
cat/KM
vmax
[sÀ1
]
[sÀ1 mÀ1
]
[mmminÀ1
]
Cys9Ala/Asp11Cys
(unmodified)
Cys9Ala/Asp11Cys-6
Cys9Ala/Asp11Cys-7
Cys9Ala/Asp11Cys-8
1.5
5.6”10À4
0.39
0.0030
1.4
1.9
1.8
4.5”10À4
5.7”10À4
4.9”10À4
0.31
0.29
0.27
0.0024
0.0031
0.0026
profile does not change substantially from the unmodified
mutant Cys9Ala/Asp11Cys to the respective bioconjugates.
The graphical representation of the bioconjugates in
Figure 3 indicates that the phenanthroline- and flavin-based
Received: June 22, 2007
Revised: August 30, 2007
Published online: October 2, 2007
Angew. Chem. Int. Ed. 2007, 46, 8597 –8600
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 8599
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Communications
[10] F. P. Seebeck, D. Hilvert, J. Am. Chem. Soc. 2003, 125, 10158 –
10159.
Keywords: bioconjugation · enzyme catalysis ·
enzyme promiscuity · hydrolases · kinetic resolution
.
[11] Y. Terao, K. Miyamoto, H. Ohta, Chem. Lett. 2007, 36, 420 – 421.
[12] a) R. Thoma, G. Obmolova, D. A. Lang, M. Schwander, P. Jenꢁ,
R. Sterner, M. Wilmanns, FEBS Lett. 1999, 454, 1 – 6; b) S.
Beismann-Driemeyer, R. Sterner, J. Biol. Chem. 2001, 276,
20387 – 20396; c) D. Lang, R. Thoma, M. Henn-Sax, R. Sterner,
M. Wilmanns, Science 2000, 289, 1546 – 1550; d) R. Sterner, B.
Hꢁcker, Chem. Rev. 2005, 105, 4038 – 4055; e) B. Hꢁcker, S.
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[13] a) Enzyme Catalysis in Organic Synthesis: A Comprehensive
Handbook, Vol. I–III (Eds.: K. Drauz, H. Waldmann), 2nd ed.,
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[14] Promiscuous reactions generally have considerably lower reac-
tion rates.[1]
[15] Mutant Cys9Ala/Asp11Cys was previously generated for other
purposes: M. T. Reetz, M. Rentzsch, A. Pletsch, A. Taglieber, F.
Hollmann, R. J. G. Mondiꢂre, N. Dickmann, B. Hꢁcker, S.
Cerrone, M. Haeger, R. Sterner, ChemBioChem, in press.
[16] Remote and close mutations in directed evolution of enzymes:
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Totowa, NJ, 2003; b) Evolutionary Methods in Biotechnology
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[17] It is interesting to note that tHisF has been shown to display
significant catalytic activity in a reaction catalyzed by a related
enzyme HisA (conversion of N’-[5’-phosphoribosyl)-form-
imino]-5-aminoimidazol-4-carboxamide ribonucleotide into 5’-
phosphoribulosyl isomer).[12c] This promiscuous reaction was
demonstrated unequivocally to occur at the natural active site of
tHisF (upper rim), which is fundamentally different from our
finding.
[1] Reviews covering various aspects of enzyme promiscuity and
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6, R91 – R105; b) S. D. Copley, Curr. Opin. Chem. Biol. 2003, 7,
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[2] Some early examples of enzyme promiscuity: a) L. A. Lokshina,
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1970, 9, 3585 – 3590; e) J. A. Peliska, M. H. OꢀLeary, Biochem-
istry 1989, 28, 1604 – 1611.
[3] a) A. Aharoni, L. Gaidukov, O. Khersonsky, S. McQ Gould, C.
Roodveldt, D. S. Tawfik, Nat. Genet. 2005, 37, 73 – 76; b) L.
Afriat, C. Roodveldt, G. Manco, D. S. Tawfik, Biochemistry 2006,
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[4] a) P. J. OꢀBrien, D. Herschlag, J. Am. Chem. Soc. 1998, 120,
12369 – 12370; b) I. Catrina, P. J. OꢀBrien, J. Purcell, I. Nikolic-
Hughes, J. G. Zalatan, A. C. Hengge, D. Herschlag, J. Am. Chem.
Soc. 2007, 129, 5760 – 5765.
[5] A. Ercan, H. I. Park, L.-J. Ming, Biochemistry 2006, 45, 13779 –
13793.
[6] a) T. Kitazume, T. Ikeya, K. Murata, J. Chem. Soc. Chem.
Commun. 1986, 1331 – 1333; b) O. Torre, I. Alfonso, V. Gotor,
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[7] M. Svedendahl, K. Hult, P. Berglund, J. Am. Chem. Soc. 2005,
127, 17988 – 17989.
[8] a) C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck,
P. Berglund, J. Am. Chem. Soc. 2003, 125, 874 – 875; b) C.
Branneby, P. Carlqvist, K. Hult, T. Brinck, P. Berglund, J. Mol.
Catal. B 2004, 31, 123 – 128.
[18] Jensen first proposed the hypothesis that organisms may
increase their survival rate under changing conditions if they
harbor promiscuous enzymes: R. A. Jensen, Annu. Rev. Micro-
biol. 1976, 30, 409 – 425. More generally Yꢂas proposed that
early enzymes must have been less specific, catalyzing classes of
reactions: M. Yꢂas, J. Theor. Biol. 1974, 44, 145 – 160.
[9] a) H. Nishino, T. Mori, Y. Okahata, Chem. Commun. 2002,
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