1. Introduction
Engineering of proteins to improve their thermostability is an area of
intensive research. Robust enzymes are required to withstand industrial
process conditions for developing biobricks for synthetic biology.
Rational design approaches to engineering thermostability require
structural information, but even with advanced computational methods it
is challenging to predict with sufficient precision to anticipate the
results of a given mutation. Directed evolution is an alternative when
structures are unavailable but it requires extensive experiments for the
screening of mutant libraries. Novel enzymes has been fueled by the
diversification of enzymes for billions of years evolution[1].
Therefore, new design method based on the co-evolution of amino acid
residues is proposed [2,3].
D-amino acids are key intermediates for pharmaceuticals and
agrochemicals [4]. L-amino acid dehydrogenases dependent on NAD(P)H
have been wildly applied for the effectively synthesis of L-amino acids.
By contrast, wide application of D-amino acid dehydrogenase (DAADH) has
not been achieved due to few DAADHs have been explored from natural
resources. Furthermore, compared with L-amino acid dehydrogenases,
DAADHs are unstable and low reaction rates[5]. Thermostability is
the bottleneck for DAADHs. Therefore, creating a stable and high active
DAADH and demonstrated that it is applicable in industry is a
challenge[6]. Designed mutations based on the structures analysis ofmeso -DAPDH and DAADH to enhance enzyme activity[7]. Cheng et
al.[8] studied the site-saturation mutagenesis of DAPDH fromSymbiobacterium thermophilum (StDAPDH) and obtain a double-site
mutant W121L-H227I, which showed improved activities towards various
sterically bulky 2-keto acids.
The development of in sillco design of enzyme reduce the
experimental costs and time[9].These interfacial interactions are
generally regulated by the key residues on the surface of motif of
enzyme. Zhu et al.[10] studied the modification of subunit
interface of Leifsonia alcohol dehydrogenase (LnADH) and yielded
the mutant T100R-S148I with thermostability with and catalytic
efficiency enhancement. Boucher et al. [11] investigated the
structural basis for the hyperthermostability of a
FN3-like protein domain from Thermoanaerobacter
tengcongensis by molecular dynamics simulations. Three arginine
residues (R23, R25, and R72), which stabilize the protein by salt
bridges were identified and mutated into alanine, which reduced melting
temperature from 97.5 to 22 °C.
How to quickly define the key interficial amino acid residues which
greatly effect stabilitiy of enzyme is crticial for protein engineering.
Co-evolution analysis can narrow down the selection range of mutated
sites, and enhance the successful rate based on the natural evolved
selection. Conservation and co-evolution analysis indicated the
interfacial interaction of domains and subunits are key factors behind
enzyme’s stability [12,13]. A limited number of co-evolved residues
located at the oligomerization interfaces, which are expected to
highlight most key sites involved in their thermostability[14]. Wang
et al.[15] redesigned the interactions of the subunit interfaces of
glycerol dehydratase from Klebsiella pneumoniae. Based on
evolutionary analysis, three co-evolved residue pairs were selected for
mutation to enhance subunits interfacial interactions. The design of
salt bridges are considered to be an easy and efficient ways to assembly
motifs, domains and subunits. Kleiner et al.[16] found that the
interdimeric interface of methionine S-adenosyltransferase fromUreaplasma urealiticum is subject to rapid evolutionary changes.
The indel loop affects the release pathway for meso -DAP and
pyruvic acid and meso -DAP[17] . Insights from co-evolution
perspective can define the key residues the interaction hotspots across
the interfacial of subunits[18]. Therefore, the novel strategy
combined with co-evolution and interfacial interaction network analysis
is developed.
D-amino acid dehydrogenase (DAADH) from Ureibacillus
thermosphaericus was selected for thermostability enhancement study
(Scheme 1). Redesign of interfacial interactions of domains and subunits
by integrating interfacial interaction network analysis with
co-evolution analysis (Fig. 1). Several novel NADPH-dependent DAADHs
were designed by multi-point mutations for interfacial interaction
enhancement. Experiments were carried out to verify the design and
reveal the mechanism of enhancement of thermostability.
Scheme.1 Reductive amination of phenylpyruvate catalyzed by
DAADH