Angewandte
Communications
Chemie
Artificial Metalloenzymes
Hot Paper
Upregulation of an Artificial Zymogen by Proteolysis
Zhe Liu+, Vincent Lebrun+, Taku Kitanosono, Hendrik Mallin, Valentin Kçhler,
Daniel Hꢀussinger, Donald Hilvert, Shu Kobayashi, and Thomas R. Ward*
Abstract: Regulation of enzymatic activity is vital to living
organisms. Here, we report the development and the genetic
optimization of an artificial zymogen requiring the action of
a natural protease to upregulate its latent asymmetric transfer
hydrogenase activity.
In a biomimetic spirit, we surmised that the activity of an
anchored catalyst precursor might be upregulated by the
action of an external trigger, ideally a natural enzyme, at
a remote position in the host protein. Such systems could form
the basis of more elaborate cross-regulated enzyme cascades
whereby the product of the ArM-catalyzed reaction inhibits
the activating enzyme.
Herein, we report the optimization of an artificial
zymogen based on the biotin-streptavidin technology
(Figure 1).
Complex biochemical cascades, which are essential to
sustain life, rely on tightly cross-regulated enzymatic process-
es. Several complementary mechanisms are used by the cell to
achieve such exquisite regulation including: 1) tuning of their
expression (e.g. transcription or translation), 2) reversible
chemical modification (e.g. phosphorylation), 3) substrate or
product inhibition, 4) reversible binding of a small molecule
distant from its active site (i.e. allosteric regulation), 5) selec-
tive proteolysis by a protease.[1–3] The latter strategy enables
organisms to spatially and temporally control the activity of
toxic enzymes that are expressed as pro-enzymes, also called
zymogens. The unraveling of their catalytic activity requires
the selective hydrolysis by a cognate protease to convert them
into their corresponding active state.
In recent years, artificial metalloenzymes (ArMs here-
after), which result from anchoring an organometallic catalyst
within a macromolecular scaffold, have emerged as an
attractive alternative to organometallic catalysts. Thanks to
their genetic encoding, well defined second coordination
sphere, and non-natural organometallic cofactor, these hybrid
catalysts combine attractive features of both homogeneous
catalysts and enzymes.[4–8] Our group recently demonstrated
that such ArMs improve the bio-compatibility of the organ-
ometallic cofactor, the protein host providing protection
against deactivation, allowing their integration into cascades
with natural enzymes.[10–12]
Figure 1. Upregulation of an artificial metalloenzyme is achieved by
proteolytic cleavage of a tripeptide. A) Structure of the biotinylated
cofactor. B) The biotinylated cofactor is activated upon coordination of
a tripeptide that is released from the C-terminus of an engineered
streptavidin (Sav=streptavidin, only one monomer depicted for
clarity; AA=amino acid).
Inspired by a publication by Whitesides, we and others
exploit the biotin–streptavidin technology for the creation of
ArMs.[13–18] In the context of upregulation, we hypothesized
that a biotinylated Cp*M moiety (M = RhIII, IrIII) might prove
versatile as 1) the [Cp*biotMCl2]2 precursor (or in combination
[*] Prof. Dr. Z. Liu,[+] Dr. V. Lebrun,[+] Dr. H. Mallin, Dr. V. Kçhler,
Dr. D. Hꢀussinger, Prof. Dr. T. R. Ward
Department of Chemistry, University of Basel
4056 Basel (Switzerland)
with wild-type streptavidin, WT Sav hereafter) shows limited
III
À
catalytic activity, both for C H activation (M = Rh ) and for
imine reduction (M = RhIII or IrIII);[19–21] 2) addition of
a suitable ligand (e.g. amidoamines, derived from natural
amino acids) leads to significant rate acceleration for the
transfer hydrogenation of imines and ketones;[19–22] and
3) embedding a Cp*Ir moiety within Sav shields the cofactor
from inhibition by other proteins, enabling enzyme cas-
cades.[10–12]
E-mail: thomas.ward@unibas.ch
Dr. T. Kitanosono, Prof. Dr. S. Kobayashi
Department of Chemistry, School of Sciences
The University of Tokyo
Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Prof. Dr. D. Hilvert
Laboratory of Organic Chemistry, ETH Zꢁrich
8093 Zꢁrich (Switzerland)
Prof. Dr. Z. Liu[+]
School of Chemistry and Chemical Engineering
Qufu Normal University
Qufu, 273165 (P.R. China)
In the context of ligand-accelerated catalysis,[23] Hilvert
et al. screened a library of tripeptides to identify ligands that
accelerate the [Cp*IrL3]-catalyzed transfer hydrogenation
(L = ClÀ or H2O). They identified glycine–glycine–phenyl-
alanine (GGF hereafter) as the tripeptide yielding the most
active complex in combination with [Cp*IrCl2]2.[19] The
tripeptide is thought to coordinate in a bidentate fashion to
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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