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336
Hybrid enzymes
Pierre Béguin
Combining structural elements belonging to different proteins is a
powerful method for generating proteins with new properties.
Progress based on detailed structural and functional analysis
enables a better integration of the elements to be fitted together
while preserving or creating functional interactions between them.
Addresses
Unité de Physiologie Cellulaire, Département des Biotechnologies,
Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France;
e-mail: [email protected]
Current Opinion in Biotechnology 1999, 10:336–340
http://biomednet.com/elecref/0958166901000336
© Elsevier Science Ltd ISSN 0958-1669
Abbreviations
Inp
ice-nucleating protein
L-lactate dehydrogenase
LDH
MDH
malate dehydrogenase
Introduction
Engineering enzymes to improve their properties is one of
the obvious goals of biotechnology. In addition, it has also
become an essential tool of research for basic protein biochemistry. In vitro DNA synthesis and recombinant DNA
technology make it possible to design and produce any
kind of polypeptide. Predicting ab initio the structure, let
alone the function of a polypeptide of known sequence,
however, still lies beyond the current capabilities of computer modelling. Hence, two strategies are left for the
creation or improvement of proteins: firstly, the creation of
random libraries; and secondly, the engineering of proteins
already existing in Nature. Even with the appearance of
powerful screening techniques, such as phage display, the
former approach is limited to very short segments, as the
number of candidates increases exponentially with the
number of residues to be varied independently. Thus, the
majority of protein engineering relies on the modification
of pre-existing proteins. For this reason, recombination of
pre-existing elements provides a very powerful tool to generate proteins with new properties. This paper will review
recent advances concerning hybrid enzymes that are most
likely to become significant for biotechnology.
As pointed out by Nixon et al. [1], the elements being
exchanged between different proteins may consist of individual residues (point mutations), secondary structure
elements, whole subdomains, or whole proteins (leading to
fusion proteins). Shorter elements are usually swapped
between homologous functional units, with a view to
retain the same basic function with modified properties:
kinetic parameters, substrate specificity, thermostability,
pH optimum, and so on. Another concept lies in the
recruiting of two or more functional units to create bi- or
multifunctional proteins.
Engineering new properties by recombining
homologous domains
Pairs of enzymes that are sufficiently related lend themselves to the construction of hybrids, either by in vivo
recombination [2] or by extension of overlapping PCR fragments in vitro [3]. Series of recombinants are then analyzed
in an attempt to identify determinants responsible for parameters such as thermostability or substrate specificity [4,5].
One problem in exchanging large segments of polypeptides
lies in the high probability that the delicate network of
interactions required for the proper structure and function
of the proteins will be perturbed. Thus, as reviewed recently by Nixon et al. [1], most attempts to modify the substrate
or cofactor specificity of the enzymes rely on the site-directed mutagenesis of one or a few residues. An interesting
concept, however, lies in the exchange of defined structural subdomains, whose proper folding is more likely to be
preserved than it would upon shuffling of arbitrary
polypeptide segments. For example, serine proteases of the
S1 family are composed of two homologous β-barrel subdomains whose interface forms the active-site cleft. The
hydrophobic core structures and the catalytic triad residues
are conserved, but the surface loops surrounding the active
site are variable, which accounts for the diverse substrate
specificity and regulatory properties of the enzymes. Thus,
S1 serine proteases are good candidates for generating new
enzymes by subdomain swapping. Hopfner et al. [6••] constructed a hybrid protease (fXYa) consisting of the
amino-terminal subdomain of the coagulation factor Xa
(fXa) and of the carboxy-terminal subdomain of trypsin.
Three-dimensional structural analysis of the hybrid showed
that the hydrophobic core elements of the interface
retained their structure with minor adjustments, but surface elements diverged more extensively from the parental
structures. Kinetic parameters of fXa, fXYa, and trypsin
were tested with a set of synthetic substrates. The recombinant protease was found to possess amidolytic activity in
the same range as the parental enzymes. In general, the
hybrid protease displayed less pronounced differences in
activity with different substrates than the parental
enzymes. Catalytic efficiencies were more similar to those
found with trypsin, in agreement with the majority of substrate binding interactions occurring with the trypsin
moiety of the hybrid. The molecular recognition profile,
however, differed significantly from that of trypsin, with a
preference inherited from fXa for substrates carrying
glycine as the penultimate residue.
Engineering bi- or multi-functional proteins
Targeting proteins with specific tags
Tagging domains can be defined as domains specifying the
distribution of a given polypeptide. They are widely used
by organisms to assign proteins to specific locations. As
Hybrid enzymes Béguin
they usually fold independently from the core protein,
they are easy to manipulate, and fusion of proteins with
specific tagging domains has now become a standard tool
for protein engineers. Vectors encoding signal peptides
have long been developed to allow secretion in various
heterologous hosts, and many are featured in catalogs of
manufacturers of molecular biology supplies. Likewise,
various commercial kits are now available to fuse polypeptides of interest with an affinity domain (maltose-binding
protein, glutathione S-transferase, cellulose- and chitinbinding domains, hexahistidine etc.) that can be easily
purified by affinity chromatography. An interesting development in this area is the construction of a tagging vector,
also commercially available, in which the carboxyl terminus of the protein of interest is linked to a chitin-binding
domain through a protein splicing element (intein) from
Saccharomyces cerevisiae [7]. The intein has been modified
so that it undergoes self-cleavage at its amino-terminus
upon incubating the fusion protein bound to chitin in the
presence of dithiothreitol or β-mercaptoethanol, which liberates the free protein.
Displaying polypeptide antigens and proteins on the surface
of bacteria has been actively pursued in the perspective of
developing live vaccines, reactions catalyzed by enzymes
immobilized on whole cells, or whole-cell adsorbents. Insertion of peptide epitopes at permissive sites of the
Escherichia coli proteins LamB and MalE has been extensively studied by the group of M Hofnung (reviewed in [8]).
A carrier polypeptide was constructed by fusing the signal
sequence and the first nine amino acids of E. coli major
lipoprotein to five transmembrane segments of the outer
membrane protein OmpA. This polypeptide was used to for
the successful display of several proteins, such as β-lactamase, a bacterial endoglucanase, a cellulose-binding
domain, and single-chain variable fragment (scFv) antibodies [9–11]. Permissive integration of foreign polypeptide
sequences within the structure of exocellular proteins is no
trivial matter, owing to the possible denaturation of the carrier protein. As an alternative, some exocellular proteins
lend themselves to constructions in which the foreign
polypeptide is fused at one end of the carrier protein.
Recently, the ice-nucleating protein (Inp) of Pseudomonas
syringae was used to display Bacillus subtilis endoglucanase
[12] or Z. mobilis levansucrase [13•] on the cell surface of
E. coli. Attachment of Inp to the cell surface appears to be
mediated via a glycosyl phosphatidylinositol anchor [14]. A
point of interest is that the length of the carrying polypeptide can be modulated by partial or total deletion of the
central region of Inp, which consists of highly repeated segments responsible for ice nucleation [12]. Display systems
have also been developed based on the use of specific
motifs involved in tethering exocellular proteins to the cell
surface. One of them is the carboxy-terminal LPXTG (single letter amino acid code, where X can be any amino acid)
motif, which anchors many exocellular proteins to the surface of Gram-positive bacteria. Upon secretion, a
transamidation occurs at the level of the threonine of the
337
motif, leading to cleavage of the precursor polypeptide and
to a peptide linkage with a free amino group of the peptide
cross-bridge in the cell wall [15,16]. This system has been
used to display fusion antigens, such as an allergen from hornet venom and a malaria blood stage antigen, on the surface
of Streptococcus gordonii [17] and Staphylococcus carnosus [18].
Preliminary evidence indicates that it would also work in
various lactic acid bacteria [19], which are non-pathogenic
and survive passage along an oral route down to the intestine. Another anchoring motif, termed the SLH domain, has
been identified in many bacterial paracrystalline surfacelayer proteins (S-layer proteins) and in several cell-bound
exocellular enzymes [20–22]. This domain, which consists
of a 60-residue segment usually reiterated threefold, appears
to bind to a polysaccharide associated with the cell wall [23].
The SLH domains of the S-layer proteins EA1 and Sap of
Bacillus anthracis were recently shown to promote in vivo
anchoring of B. subtilis levansucrase to the cell surface of
B. anthracis [24•].
Fusion polypeptides have also been considered as a
method to target proteins to specific animal cells. The construction of toxic fusion proteins attacking specific
disease-causing cells has been actively pursued (reviewed
in [25]). In addition, the nontoxic carboxy-terminal fragment of tetanus toxin has been fused to β-galactosidase to
label motoneurons and connected neurons by retrograde
transport [26]. Fusion to superoxide dismutase enabled
delivery and internalization of the enzyme by neuronal
cells in culture [27]. The latter work opens up the possibility of treating neurological diseases involving free
radical damage to neurons, such as amyotrophic lateral
sclerosis. It remains to be demonstrated, however, that the
superoxide dismutase-tetanus toxoid hybrid actually protects neurons against such damage.
Tagging sequences can also be used to address enzymes to
specific DNA sequences. For example, Kim et al. [28] have
explored the possibility of creating new restriction
enzymes by fusing zinc fingers that bind to specific
sequences with the cleavage domain of Fok I endonuclease. The results showed that specific cleavage did occur,
although the hybrid enzymes were much less active than
wild-type Fok I. The cleavage specificity was variable,
some zinc fingers yielding more spurious cleavage at secondary sites than others. DNA recognition sites of RNA
polymerase have also been manipulated by creating new
hybrid subunits. A hybrid σ subunit was constructed by
combining the amino-terminal region of the major E. coli σ
subunit (σ70) with the carboxy-terminal end of σ32, which
recognizes heat shock promoters. The hybrid promoted
transcription from a chimeric promoter composed of the
σ32 –35 consensus and of the σ70 –10 consensus sequence
[29]. Likewise, Mencía et al. [30] made an RNA polymerase responsive to a B. subtilis transcriptional activator
by replacing the wild type E. coli α subunit with a hybrid
in which the carboxy-terminal domain was replaced by the
corresponding domain of the α subunit from B. subtilis.
338
Protein technologies and commercial enzymes
timodule complexes involved in polyketide and nonribosomal peptide synthesis, which yield many
compounds with antibiotic or pharmacological activity.
In both cases, synthesis proceeds by the sequential addition of specific elements, which is performed by
specialized modules. Synthetic modules are part of multimodular polypeptides, which themselves form large
synthase complexes responsible for synthesis of the full
polyketide or peptide chain. Thus, the order and nature
of the modules determines the structure of the final
product. Shuffling, deleting or modifying specific modules offers the opportunity for generating an almost
unlimited variety of compounds, and the field is currently the focus of intense research activity (for reviews see
[33,34•]).
Figure 1
Catalytic domains
Dockerin
domains
Cohesin
domains
Current Opinion in Biotechnology
Modelling of artificial complexes having a defined stoichiometry and
topology based on cohesin and dockerin domains with different
recognition specificities. Cohesin domains are linked together in a defined
order into a multimodule scaffolding polypeptide. Catalytic domains to be
integrated within the complex are linked to appropriate dockerin domains
in order to interact with the various cohesin domains of the scaffolding
polypeptide. Linker segments are indicated by black bars.
A very important development based on a tagging strategy
is the two-hybrid system used for the screening of protein–protein interactions. In the original version, protein
association is detected by reconstituting an active transcriptional activator in yeast [31]. More recently, a similar
system has been developed based on fusions with Bordetella pertussis adenylate cyclase fragments. Association of the
fragments generates sufficient cyclase activity to be monitored by the activity of a reporter gene whose expression is
dependent on cAMP. The advantage is that the system is
functional in E. coli. In addition, because cAMP is diffusible the interacting partners need not be located in the
compartment where transcription occurs [32••].
Natural and artificial systems for grouping
different enzymes
Nature provides many examples of bi- or multifunctional enzyme systems performing consecutive reactions in
various metabolic pathways. Adequate spatial arrangement of catalytic subunits plays a critical role by
ensuring that reactions occur in the proper order, and by
speeding up the process owing to efficient channeling of
intermediate products from one reaction center to the
next one. Among the most evolved systems are the mul-
Bifunctional proteins constructed de novo to perform coupled reactions were investigated in the late 1980s and early
1990s by Mosbach, Bülow and their co-workers [35–37].
Several fusion proteins were tested, including hybrids of
β-galactosidase and galactose dehydrogenase [35], galactose dehydrogenase and bacterial luciferase [36], and
malate dehydrogenase and citrate synthase [37]. The
steady-state activity of the coupled enzyme systems measured for the coupled reactions was increased up to
2–3-fold as compared to the separated enzymes [35], and
the pre-steady state lag was reduced up to 4–6-fold [37].
The enhancement of activity observed upon coupling the
reaction centers was maximal when the activity of the first
enzyme was limiting owing to non-optimal pH conditions
or low concentrations of enzyme or substrate [35,36].
Thus, the effect of coupling on activity was not overwhelming, but nonetheless significant. Better results
would probably be expected if it were possible to optimize
the position of the catalytic centres relative to one another.
As opposed to fusion proteins, hybrid multiprotein complexes offer the possibility to assemble several catalytic centres
without synthesizing huge polypeptides. In this respect, the
cellulolytic complex, or cellulosome, produced by the bacterium Clostridium thermocellum, lends itself to manipulation
with remarkable versatility. The cellulosome consists of up
to 25 different enzymes that attack cellulose and associated
plant cell-wall polysaccharides. Each of the catalytic subunits
is anchored by means of a conserved domain, termed the
dockerin domain, to a large scaffolding protein termed CipA.
CipA itself consists of a set of nine highly similar receptors,
termed cohesin domains, which bind the dockerin domains
borne by the catalytic subunits (for a review see [38]). Dockerin domains can be fused to foreign polypeptides,
mediating the integration of arbitrarily chosen proteins into
artificial complexes [39,40]. Cohesin domains comprised in
the sequence of CipA have very similar binding specificities,
however, precluding the arrangement of the polypeptides to
be associated in a chosen order and stoichiometry. A possible
solution lies in the construction of artificial scaffolding
polypeptides carrying cohesin domains of differing specificity, whose number and order could be chosen arbitrarily. The
Hybrid enzymes Béguin
polypeptides to be integrated in the complex would accordingly be fused to dockerin domains of appropriate specificity
(Figure 1). Indeed, a heterodimer consisting of two cohesin
domains with distinct recognition specificities was shown to
mediate the formation of complexes integrating two different polypeptides provided with appropriate dockerin
domains [41]. The versatility of the system could be further
increased by including cohesin and dockerin domains from
related organisms, such as Clostridium cellulolyticum, which
have been demonstrated to possess a different recognition
specificity [42]. Alternatively, new types of dockerin–cohesin
interactions could be designed and created by site-directed
mutagenesis when the structure of cohesin–dockerin complexes is elucidated.
Engineering allosteric interactions between artificially
combined domains
Most of the bifunctional proteins described above are
made up of domains connected side by side, which presumably retain the capacity to fold independently and
possess a considerable degree of conformational freedom
relative to one another. Although this ensures that the
domains are likely to remain functional within the hybrid,
it also means that the spatial orientation of their active sites
relative to one another is loose and cannot be optimized.
Furthermore, the possibility of engineering allosteric interactions would offer interesting opportunities, for example,
by modulating the activity of enzymes, which could then
act as biosensors. One way to achieve a closer interaction
between two domains is to graft one of them at a permissive site within the sequence of the second one.
Accordingly, β-lactamase was fused to the maltodextrinbinding protein MalE, either by insertion at various sites
within MalE or by fusion at the carboxyl terminus of the
polypeptide chain. Insertion after residues 133 and 303,
which were known to accommodate small peptides or
deletions, resulted in the secretion into the periplasm of
fusion proteins capable of transporting maltose and
hydrolyzing β-lactams. The same was true of the carboxyterminal fusion, but the internal fusions displayed two
additional properties: firstly, they were less sensitive to
degradation by endogenous proteases; and secondly, addition of maltose stabilized the activity of the β-lactamase
domain against denaturation by urea, indicating a true
allosteric interaction between the two domains [43].
The possibility of developing a sensor system based on the
modulation of alkaline phosphatase activity was explored
by Brennan et al. [44]. Epitopes of HIV type I and of
hepatitis C virus were grafted within the sequence of alkaline phosphatase, and the activity of the recombinant
enzyme was tested in the presence or absence of antibodies directed against these epitopes. When epitopes were
inserted into wild-type alkaline phosphatase, addition of
specific antibodies reduced by 14–17% the activity of the
hybrids. Antibodies increased the activity of the fusion
proteins up to 2–3-fold when the epitopes were inserted
339
into two mutants of alkaline phosphatase that displayed
increased flexibility and lower thermal stability.
Finally, allosteric activation can also be manipulated in
multimeric complexes. For example, the substrate specificity of L-lactate dehydrogenase (LDH), which is a
tetrameric enzyme subject to homotropic activation, can
be changed into malate dehydrogenase (MDH) by a single
site mutation. Unlike LDH, the resulting MDH does not
bind low concentrations of oxamate, a non-reactive analog
of pyruvate. Fushinobu et al. [45•] constructed and purified
various heterotetramers containing both LDH and MDH.
In such hybrids, MDH activity was enhanced in the presence of oxamate, which acted like a homotropic allosteric
effector by binding to the LDH sites of the complexes.
Conclusions
Constructing hybrid polypeptides is a major aspect of protein engineering and covers a very wide range of actual or
potential applications. So far, the field has been largely
concerned with the mere physical connection of protein
segments without paying much attention to the spatial
integration of the parts composing the fusion polypeptides.
This approach brought about significant success, particularly in the case of tagging strategies, for which tagging
domains do not normally need to interact specifically with
the rest of the protein. It is probable, however, that more
sophisticated designs, based on a detailed knowledge of
the structure and function of the elements to be combined,
will become increasingly popular. They will be required to
develop the full potential of hybrid proteins, particularly
for constructing integrated multienzyme complexes or for
manipulating the regulation of enzyme activity.
References and recommended reading
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have been highlighted as:
• of special interest
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anchoring of functional fusions between the SLH motifs of the
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45. Fushinobu S, Ohta T, Matsuzawa H: Homotropic activation via the
•
subunit interaction and allosteric symmetry revealed on analysis
of hybrid enzymes of L-lactate dehydrogenase. J Biol Chem 1998,
273:2971-2976.
A recent paper demonstrating how the substrate specificity of a multimeric enzyme can be modified while retaining sensitivity to the original
allosteric effector.