\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Design of Motifs Interfacial Interactions by Co-evolved Analysis of
D-amino Acid Dehydrogenase for Stability Enhancement}



\author[1]{Zhehui Ji}%
\author[1]{Qian Zhang}%
\author[1]{Lingxuan Duan}%
\author[1]{Lingling Dong}%
\author[1]{Shizhen Wang}%
\affil[1]{Xiamen University}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
To design D-amino acid dehydrogenase (DAADH) for enhanced stability, the
interactions of the subunit interfaces of DAADH were analyzed.
Interaction network analysis of DAADH indicated that there are only weak
interactions between the A and B subunits. Several co-evolved residue
pairs were selected for mutation to enhance interfacial interactions of
subunits, and 11 designed MDHs were obtained. DA06 and DA11 were
selected for experimental verification for their salt bridges are 1.4
and 1.2-fold of that of DAwild, respectively. DA11 can maintain 93\%
activity in 80, while it was only 40\% for DAwild. Thermostabiliy study
indicated the half-life of DA11 was 2-fold of DAwild. Molecular dynamics
simulations revealed that the extraordinary stability of the DA11 was
due to the formation of extra interfacial salt bridges. The paper
provided a strategy of mutations outside the active site of enzyme by
co-evolutionary analysis which can reduce the effect of the
activity-thermostability trade-off.%
\end{abstract}%



\sloppy


\textbf{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
of\emph{meso} -DAPDH and DAADH to enhance enzyme activity{[}7{]}. Cheng
et al.{[}8{]} studied the site-saturation mutagenesis of DAPDH
from\emph{Symbiobacterium thermophilum} ~ (StDAPDH) and obtain a
double-site mutant W121L-H227I, which showed improved activities towards
various sterically bulky 2-keto acids.

The development of \emph{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~\emph{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
FN\textsubscript{3}-like protein domain from \emph{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 \selectlanguage{ngerman}°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 \emph{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
from\emph{Ureaplasma urealiticum} is subject to rapid evolutionary
changes. The indel loop affects the release pathway for~\emph{meso} -DAP
and pyruvic acid and~\emph{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 \emph{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.

\textbf{Scheme.1} Reductive amination of phenylpyruvate catalyzed by
DAADH

\textbf{2. Materials and methods}

\emph{2.1. Structures}

The X-ray diffraction structure of D-amino acid dehydrogenase
of\emph{Ureibacillus thermosphaericus} was retrieved from the Protein
Data Bank (PDB). In this work, the crystal structure of the DAADH (PDB
code 5gz6) was employed for amino acid residues interaction analysis and
molecule dynamic simulation.

\emph{2.2. Amino acid residues interaction analysis}

The web server of Protein Contacts Atlas\textbf{(}
http://www.mrc-lmb.cam.ac.uk/pca/\textbf{)} was applied for analysis of
non-covalent contacts, such as hydrogen bonds, hydrophobic interactions,
cation-pi interactions, salt bridges, and etc. The default threshold of
the atoms' distance for interaction calculations is set as 0.5 Å.
Multiple non-covalent contacts at different scales of structure, namely,
amino acid residues, secondary structure, subunits, and entire complexes
were analyzed{[}19{]}. Protein side-chain networks (PScN) is then
constructed by setting amino acid residues as nodes. The edges are
constructed between the nodes based on non-covalent interactions,
side-chain interaction between them.

\emph{2.3. Co-evolution and conservation and analysis}

The calculation of co-evolved residue pairs was based on Evcoupling
webserver. Evcoupling can calculate co-evolution score for each residues
in the corresponding protein family{[}20{]}. The co-evolution score
reflect a network of interactions between residue pairs based on the
evolutionary information in the family{[}21{]}. Conservation analysis
was carried out by the ConSurf webserver
(http://consurf.tau.ac.il/2016/) with HMMER E-value was set as 0.0001.

\emph{2.4. Thermodynamic parameters calculation}

Thermodynamic parameters, such as change of
enthalpy(\selectlanguage{greek}Δ\selectlanguage{english}H\textsubscript{m}), change of Gibbs free energy of stability
(\selectlanguage{greek}Δ\selectlanguage{english}G\textsubscript{r}), change of heat capacity (\selectlanguage{greek}Δ\selectlanguage{english}C\textsubscript{p}) and
melting temperature (T\textsubscript{m}) were calculated by the SCooP
webserver, which is freely available at~http://babylone.ulb.ac.be/SCooP.

\emph{2.5. Chemicals}

Kanamycin and isopropyl-\selectlanguage{greek}β-\selectlanguage{english}D-thiogalactopyranoside (IPTG) are purchased
from Trans-Gen Biotech (Shanghai, China). NADPH and phenylpyruvate are
purchased from Sigma Chemical Company (Tianjing, China). The ionic
liquids (99\% purity) are purchased from Sigma Chemical Company
(Tianjing, China). All other chemicals are analytical grade and
purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).

\emph{2.6. Enzyme expression and purification}

The daadh gene encoding the wild type D-amino acid dehydrogenase (DAADH)
was synthesized by Sangon Biotech (Shanghai) Co. Ltd. DAADH originates
from Ureibacillus thermosphaericus (PDB ID: 5gz6) was selected. An
expression vector for DAADH\_5gz6 with polypeptide linker (amino acid
sequence (RTHRK)4) were constructed by amplifying the DAADH\_5gz6 gene
fragment infusion by PCR.

The amplified gene fragment was double-digested with NdeI and XhoI and
ligated cloned into plasmid pET-28a(+), with the expression vector
E.coli BL21(DE3)/pET28a. The plasmid extraction and gel recovery kit
(Takara) was used. Cells were cultivated at 37 \selectlanguage{ngerman}°C and 200 r/min in 10 mL
of LB medium containing 50 \selectlanguage{greek}μ\selectlanguage{english}g/L kanamycin until the optical density (OD)
at 600 nm reached 0.6-0.8. Then, gene expression was induced by adding
isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-thiogalactopyranoside (IPTG) for an additional 12 h at 25
\selectlanguage{ngerman}°C.

The harvested cells were washed twice with 20 mM Tris-HCl buffer (pH
7.0) and suspended in a buffer of 50 mM
Na\textsubscript{2}HPO\textsubscript{4} (pH 7.0). Suspended cells were
disrupted by ultrasonication and centrifuged at 100,000×g for 1 h. The
supernatant was purified using a Ni\textsuperscript{2+}-NTA column
(Seven sea biotech, Shanghai, China) to obtain samples for activity
assay.

\emph{2.7. Enzyme activity assay}

The activities of DAADHs were determined in
NH\textsubscript{4}Cl-NH\textsubscript{3}·H\textsubscript{2}O buffer
(200 mM, pH 9.5) with phenylpyruvate and NADPH. Enzyme activity was
measured by detecting the NADPH concentration at 340 nm by Infinite 200
PRO spectramicroplate reader (TECAN). The initial velocities was
calculated as consumption rate of NADPH when the conversion rate was
below 5\%. The amount of enzyme consumed for generation or consumption
of 1 \selectlanguage{greek}μ\selectlanguage{english}mol of NADPH per minute was defined as one unit of enzyme
activity. Specific activity (U/mg) of DAADH is defined as the ratio of
enzyme activity (U/mL) to enzyme concentration (mg/mL). Each data was
obtained by five parallel experiments.

D-phenylalanine was analyzed by HPLC. HPLC analysis with standard
solution of L-phenylalanine and D-phenylalanine showed that the peak
time of L-phenylalanine was 25 min, and the peak time of D-phenylalanine
was in 33 min. HPLC detection condition for D/L phenylalanine was as
follow: Chromatographic column: Philomon Chirex 3126 (D)-Penicillamine
250 nm*4.6 nm, 5 \selectlanguage{greek}μ\selectlanguage{english}m; mobile phase, 5\% (v/v) acetonitrile and 95\% (v/v)
2.0 mM, CuSO\textsubscript{4} aqueous solution; flow rate, 0.8 mL/min;
detection wavelength, 254 nm{[}22{]}.

\textbf{3. Result and discussions}

\emph{3.1. Interfacial interaction analysis}

In an effort to redesign DAADH for improved stability, interfacial
interactions of A and B subunits of DAADH from \emph{Ureibacillus
thermosphaericus} (DAwild) were analyzed by Protein Contacts Atlas.
Amino acid residues interaction analysis indicated that there are only
weak interfacial interactions between the A and B subunits, which are
only one salt bridges and 12 hydrogen bonds. The salt bridge was form by
GLU136 of A subunit and LYS316 of B subunit with the distance of 3.177 \selectlanguage{ngerman}Å
(Fig. 2A). Interactions heatmap of Helix 15 and 16 of A subunit and
Helix 23 of B subunit were investigated and key residues were
highlighted (Fig. 2B). Therefore, the interfacial interactions of
subunits and motifs can be further improved to enhance the stability of
the enzyme {[}23{]}.

\emph{3.2. Define key residues by conservation and coevolution analysis}

Conservation scores were calculated for each residue of DAADH using
ConSurf webserver (Fig. S1). Conservation analysis indicated that key
residues which maintain the interfacial interactions are more conserved
than others during the evolution (Table 1) {[}24{]}.

Evolutionary coupling reflect the network of interactions between
residue pairs based on their evolutionary information in the
family{[}13{]}. Several co-evolved residues pairs are identified by
co-evolution analysis of dimeric interfacial interactions (Table 2).
Co-evolutionary analysis of protein residues can reveal the underlying
remote interactions of amino acid residues (Fig. 3A). Therefore, several
co-evolved residues pairs which are selected for mutation to enhance the
subunits interaction (Fig. 3B).

\emph{3.3. Design of interfacial interactions}

Double mutations of V137I-S314L, F306H-G311I, L134W-F310I were designed
to enhance the hydrogen bonds and salt bridges, which aim to promote
structure rigidity of enzyme. Calculation of mutation energy was carried
out by the webserver Calculate Mutation Energy of Discovery Studio 2016
Client. All mutation energy of double mutated enzymes was negative,
which indicated the enhanced thermostability. Therefore, positive
mutations were further combined and the designed enzymes DA06 to DA11
were obtained. None of mutated sites is located within active sites,
which would not arise activity change from binding substrate. Mutation
energy for the combined mutations indicated the cooperative effect on
the improvement of thermostability (Table 2).

Thermodynamic parameters of all designed enzymes were calculated by
Scoop and were listed in Table 3 {[}25{]}. Parameters such as change in
enthalpy (\selectlanguage{greek}Δ\selectlanguage{english}H\textsubscript{m}), change in heat capacity
(\selectlanguage{greek}Δ\selectlanguage{english}C\textsubscript{p}), melting temperature (T\textsubscript{m}) and
difference in Gibbs free energy of stability (\selectlanguage{greek}Δ\selectlanguage{english}G\textsubscript{r}), were
calculated. These thermodynamic parameters which have important
implications for enzyme thermostablility{[}26{]}.

\emph{3.4. Energy and interaction analysis of designed enzymes}

Molecular dynamics simulations (MDS) of designed enzyme were carried
out. Energy decomposition analysis of mutation was studied by data
obtained from MDS. Co-evolved residues pairs were mutated in order to
form new and strong salt bridges. Electrostatic interaction energy and
total interaction energy was calculated for further selection of mutated
pairs (Table 4), which indicated that electrostatic interaction energy
contribute to the most part of interaction energy. Buried interactions
network can enhance interfacial interactions of subunits and enzyme
stability {[}27{]}.

The benefits of considering dynamic conformation change for enzyme
design can also enable engineering enzymes from the prospect of both
structure and function{[}28{]}. The structure of the enzyme undergoes
dynamic conformational adjustments with increasing temperature,
resulting in gradual loosening of the unstable secondary structure and
consequently disfolding. The introduction of interfacial hydrogen bonds
contributes to the thermostability of the enzyme{[}29{]}. Energy
provided by each hydrogen bonds is about 0.6 kcal/mol {[}30{]}. MDS also
indicated that the average number of hydrogen bonds of wild-type DAwild
is 108. Therefore, there are 8 extra hydrogen bonds of DA11 than DAwild,
which provided extra energy barrier for unfolding (Table 5).

The average number of salt bridges of DAADHs were calculated by MDS
result with 0.1 M NaCl. Salt bridges networks play an important role in
maintain the rigidity of the three-dimensional structure of enzyme.
Formation of more salt bridges inside the protein molecular is
beneficial to maintain the structural stability{[}31{]}. The average
number of salt bridges of DA06 and DA11 are 1.4 and 1.2-fold of that of
DAwild, respectively. This result indicated that increased salt bridges
and hydrogen bonds enhanced structural rigidity and improved
thermostability.

Therefore, DA06 and DA11 were selected for further experiment study. The
formation of a large number of non-covalent interactions within the
modified enzyme is beneficial to the thermostability, which has been
strengthened by multipoint mutations.

\emph{3.5. Experimental verification by mutation}

Activity of mutations were tested. The activity of DAwild and DA11 at 25
\selectlanguage{ngerman}°C were 2.46 and 2.47 U/mg, respectively, while the activity of DA06 was
only 0.27 U/mg. This result indicated the DA11 of interfacial
interactions would not affect the enzyme activity. Normally, engineering
enzyme with improved the rigidity for thermostability enhancement cause
the decrease of activity. Multi-site mutation for enzyme activity
improvement can affect the stability, which is a compromise between
enzyme activity and stability {[}28{]}.

Interaction analysis indicated that the 12 mutations of DA06
(L133W-V136I-F305C-F309I-G310W-S313Q) led to the formation of salt
bridges between 346LYS-408GLU, 349LYS-375GLU, 349LYS-408GLU (Table S2).
New formed salt bridges elucidated the mechanism of a key amino acid
residue mutation enhanced thermostability {[}17{]}. However, to much
extra salt bridges and hydrogen bonds of DA06 decreased enzyme activity
due to the effect of activity-stability trade-off by enhanced the
rigidity{[}32{]}.

Effect of pH on DAADHs was studied. Among them, DA11 is with the widest
adaptable pH range, especially in alkaline solutions (Fig. 4a).
Catalytic properties the designed enzyme were verified. The effect of
temperature on DAwild, DA06 and DA11 activity was studied in the range
of 30 to 80 °C. The optimal temperature for DA06 and DA11 was 60 °C,
while it was only 50 °C for DAwild (Fig. 4b). DA11 can maintain 93\%
activity in 80 °C, while it was only about 40\% for DAwild. This result
indicated the interifical mutations of subunits enhanced thermostability
of DA11 without decreasing the activity.

\emph{3.6. Thermostability study}

Thermostability of DA11 and DAwild was studied under 50 \selectlanguage{english} (Fig. 5). The
deactivation kinetic parameters was obtained, and the half-life of DA11
was 11.2 h, almost 2-fold of DAwild (Table S1). Thermostability of DA11
was further studied under 60, 70, 80 \selectlanguage{english} (Figure S2). DA11 retained 52\%
activity under 80 \selectlanguage{english} after 10 h. The result indicated interfacial
interactions enhancement greatly improved thermostability. Mutations of
DA11 on the subunit interface were marked in Fig. 6. MDS result
indicated the salt bridges formed between Lys300-Glu482, Lys341-Asp407,
Arg324-Asp407, Lys346-Asp410, Lys439-Glu375, Arg351-Asp410 indicated the
enhanced thermostability by interfacial interactions of subunits of
DA11(Table S3). Therefore, co-evolutionary analysis are sufficient to
identify higher-than-pairwise mutation patterns of DAADH, which can both
increase the activity and thermostability.

\emph{3.7. Kinetic study of DAADH}

Kinetic parameters of the DAwild and DA11 were calculated by Hanes-Woolf
method within the phenylpyruvate concentration range of 0.625-10 mM, as
shown in Fig. S3. V\textsubscript{max} of DAwild and DA11 are all 0.5
mM/min, while DA11 showed a decreased K\textsubscript{m} value as 1.00
mM (Table 6). This result indicated that the DA11 increased the affinity
with substrate, and further improved enzyme catalyzed efficiency.
Interfacial mutation which are outside the active sites can improved
stability without decrease the enzyme activity, which can reduce the
trade-off effect of activity and thermostability.

\emph{3.8. Effect of ionic liquids on the enzyme activity}

Ionic liquids (ILs) is with industrially attractive advantages for
biocatalysis due to their tunable chemical properties. Several ionic
liquids ({[}EMIM{]}BF\textsubscript{4}, {[}BMIM{]}Cl,
{[}BMIM{]}BF\textsubscript{4}) were selected and added into the reaction
system with final concentration as 5 mM (Fig. S4). The activity of DA11
in {[}BMIM{]}BF\textsubscript{4} ionic liquid was 97.8\%, while
{[}EMIM{]}BF\textsubscript{4} and {[}BMIM{]}Cl reduced the relative
enzyme activity of DA11. It is reported that
{[}BMIM{]}\textsuperscript{+} cations can strip off essential water
molecules from the enzyme surface, which cause the decreased enzyme
activity {[}33{]}. Ionic liquids also can affect enzyme activity through
direct interaction with the enzyme and by altering the microenvironment
of enzyme. Due to the conductivity and hydrophobicity, ILs are widely
applied for electrode modification to obtain high heterogeneous electron
transfer rates {[}34{]}. Therefore, ILs tolerant DA11 is promising for
its application in biosensor.

\textbf{4. Conclusion}

To improve the thermostability of DAADH, several co-evolved interfacial
residue pairs were identified. Based on the amino acid residue
interaction network and co-evolutionary analysis, those residues were
mutated for redesign the interfacial interaction of subunits. Eleven
novel DAADHs were obtained. Mutations induced new salt bridges and
hydrogen bonds. The average number of salt bridges of DA06 and DA11 are
1.4 and 1.2-fold of that of DAwild, respectively. Thermostability study
indicated the half-life of DA11 was 2-fold of DAwild. MDS was applied to
uncover the mechanism of enhanced thermostability from molecular level.
These findings suggest that the dimer interface motifs are essential for
the compactness and stability of DAADHs and may illuminate design based
on evolution in other enzyme families.

\textbf{Acknowledgements}

This work was supported by the National Natural Science Foundation of
China (No. 22078273, 21776233).

\textbf{Abbreviations}

\textbf{DAADH,} D-amino acid dehydrogenase; \textbf{MDS,} Molecular
dynamic simulation; \textbf{RMSD,} Root mean-square
deviations;\textbf{RMSF,} Root mean-square fluctuations; \textbf{PDB,}
Protein Data Bank.

\textbf{Conflict of interest statement}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{References}

\textbf{1.} Spence MA, Kaczmarski JA, Saunders JW, Jackson CJ. Ancestral
sequence reconstruction for protein engineers. Current Opinion in
Structural Biology. 2021 AUG 2021;69:131-141.

\textbf{2.} Thomson RES, Carrera-Pacheco SE, Gillam EMJ. Engineering
functional thermostable proteins using ancestral sequence
reconstruction. The Journal of Biological Chemistry. 2022 Aug 27 (Epub
2022 Aug 27) 2022;298(10):102435-102435.

\textbf{3.} Johansson KE, Lindorff-Larsen K. Structural heterogeneity
and dynamics in protein evolution and design. Current Opinion in
Structural Biology. 2018 FEB 2018;48:157-163.

\textbf{4.} Hayashi J, Seto T, Akita H, et al. Structure-based
engineering of an artificially generated NADP(+)-dependent d-amino acid
dehydrogenase. Applied and Environmental Microbiology. 2017 JUN
2017;83(11).

\textbf{5.} Pollegioni L, Rosini E, Molla G. Advances in enzymatic
synthesis of d-amino acids. Internatoinal Journal of Molecular Sciences.
2020 MAY 2020;21(9).

\textbf{6.} Prier CK. Enzymes for amino acid synthesis by design. Nature
Catalysis. 2021 MAY 2021;4(5):348-349.

\textbf{7.} Akita H, Hayashi J, Sakuraba H, Ohshima T. Artificial
thermostable d-amino acid dehydrogenase: creation and application.
Frontiers in Microbiology. 2018 AUG 3 2018;9.

\textbf{8.} Cheng X, Chen X, Feng J, Wu Q, Zhu D. Structure-guided
engineering of meso-diaminopimelate dehydrogenase for enantioselective
reductive amination of sterically bulky 2-keto acids. Catalysis Science
\& Technology. 2018 OCT 7 2018;8(19):4994-5002.

\textbf{9.} Wilding M, Scott C, Warden AC. Computer-guided surface
engineering for enzyme improvement. Scientific Reports. 2018 AUG 10
2018;8.

\textbf{10.} Zhu L, Song Y, Chang C, et al. Engineering leifsonia
alcohol dehydrogenase for thermostability and catalytic efficiency by
enhancing subunit interactions. Chembiochem. 2021 NOV 16
2021;22(22):3178-3183.

\textbf{11.} Boucher L, Somani S, Negron C, et al. Surface salt bridges
contribute to the extreme thermal stability of an FN3-like domain from a
thermophilic bacterium. Proteins- Structure Function and Bionformatics.
2022 JAN 2022;90(1):270-281.

\textbf{12.} Arcus VL, van der Kamp MW, Pudney CR, Mulholland AJ. Enzyme
evolution and the temperature dependence of enzyme catalysis. Current
Opinion in Structural Biology. 2020 DEC 2020;65:96-101.

\textbf{13.} Hockenberry AJ, Wilke CO. Evolutionary couplings detect
side-chain interactions. Peerj. 2019 JUL 8 2019;7.

\textbf{14.} Sun Z, Liu Q, Qu G, Feng Y, Reetz MT. Utility of B-Factors
in Protein Science: Interpreting rigidity, flexibility, and internal
motion and engineering thermostability. Chemical Reviews. 2019 FEB 13
2019;119(3):1626-1665.

\textbf{15.} Zhang Y, Ren H, Wang Y, Chen K, Fang B, Wang S. Bioinspired
immobilization of glycerol dehydrogenase by metal ion-chelated
polyethyleneimines as artificial polypeptides. Scientific Reports. 2016
APR 7 2016;6.

\textbf{16.} Kleiner D, Shmulevich F, Zarivach R, et al. The
interdimeric interface controls function and stability of ureaplasma
urealiticum methionine s-adenosyltransferase. Journal of Molecular
Biology. 2019 DEC 6 2019;431(24):4796-4816.

\textbf{17.} Huang L, Ma J, Sang J, et al. Enhancing the thermostability
of phospholipase d from Streptomyces halstedii by directed evolution and
elucidating the mechanism of a key amino acid residue using molecular
dynamics simulation. International Journal of Biological Macromolecules.
2020/12/01/ 2020;164:3065-3074.

\textbf{18.} Copley SD. Setting the stage for evolution of a new enzyme.
Current Opinion in Structural Biology. 2021 AUG 2021;69:41-49.

\textbf{19.} Kayikci M, Venkatakrishnan AJ, Scott-Brown J, Ravarani CNJ,
Flock T, Babu MM. Visualization and analysis of non-covalent contacts
using the protein contacts atlas. Nature Structural \& Molecular
Biology. 2018 FEB 2018;25(2):185-+.

\textbf{20.} Oteri F, Nadalin F, Champeimont R, Carbone A. Bis2analyzer:
a server for co-evolution analysis of conserved protein families.
Nucleic Acids Research. 2017 JUL 3 2017;45(W1):W307-W314.

\textbf{21.} Ashkenazy H, Abadi S, Martz E, et al. Consurf 2016: an
improved methodology to estimate and visualize evolutionary conservation
in macromolecules. Nucleic Acids Research. 2016 JUL 8
2016;44(W1):W344-W350.

\textbf{22.} Wang S, Duan L, Jiang L, Liu K, Wang S. Assembly of peptide
linker to amino acid dehydrogenase and immobilized with metal-organic
framework. Journal of Chemical Technology and Biotechnology. 2022 MAR
2022;97(3):741-748.

\textbf{23.} Tajik S. Topological analysis of high temperature enzyme
activity. Biophysical Journal. 2022/02/11/ 2022;121(3, Supplement
1):320a.

\textbf{24.} Sfriso P, Duran-Frigola M, Mosca R, Emperador A, Aloy P,
Orozco M. Residues coevolution guides the systematic identification of
alternative functional conformations in proteins. Structure. 2016 JAN 5
2016;24(1):116-126.

\textbf{25.} Pucci F, Kwasigroch JM, Rooman M. Scoop: an accurate and
fast predictor of protein stability curves as a function of temperature.
Bioinformatics. 2017 NOV 1 2017;33(21):3415-3422.

\textbf{26.} Van der Kamp MW, Prentice EJ, Kraakman KL, Connolly M,
Mulholland AJ, Arcus VL. Dynamical origins of heat capacity changes in
enzyme-catalysed reactions. Nature Communications. 2018 MAR 21 2018;9.

\textbf{27.} Vijayabaskar MS, Vishveshwara S. Interaction energy based
protein structure networks. Biophysical Journal. 2010 DEC 1
2010;99(11):3704-3715.

\textbf{28.} Agarwal PK, Bernard DN, Bafna K, Doucet N. Enzyme dynamics:
looking beyond a single structure. Chemcatchem. 2020 OCT 6
2020;12(19):4704-4720.

\textbf{29.} Zhang W, Zhang Y, Huang J, et al. Thermostability
improvement of the d-allulose 3-epimerase from dorea sp CAG317 by
site-directed mutagenesis at the interface regions. Journal of
Agricultural and Food Chemistry. 2018 JUN 6 2018;66(22):5593-5601.

\textbf{30.} Liu Y, Luo G, Huu Hao N, Guo W, Zhang S. Advances in
thermostable laccase and its current application in lignin-first
biorefinery: A review. Bioresource Technology. 2020 FEB 2020;298.

\textbf{31.} Chan C-H, Yu T-H, Wong K-B. Stabilizing salt-bridge
enhances protein thermostability by reducing the heat capacity change of
unfolding. Plos one. 2011 JUN 24 2011;6(6).

\textbf{32.} Yu H, Dalby PA. Exploiting correlated molecular-dynamics
networks to counteract enzyme activity-stability trade-off. Proceedings
of the National Academy of Sciences of the United States of America.
2018 DEC 26 2018;115(52):E12192-E12200.

\textbf{33.} Cui H, Stadtmuller THJ, Jiang Q, Jaeger K-E, Schwaneberg U,
Davari MD. How to engineer organic solvent resistant enzymes: insights
from combined molecular dynamics and directed evolution study.
Chemcatchem. 2020;12(16):4073-4083.

\textbf{34.} Badgujar KC, Badgujar VC, Bhanage BM. Recent update on use
of ionic liquids for enzyme immobilization, activation, and catalysis: a
partnership for sustainability. Current Opinion in Green and Sustainable
Chemistry. 2022 AUG 2022;36.

\textbf{Hosted file}

\verb`Data availability statement.docx`  available at \url{https://authorea.com/users/592947/articles/628214-design-of-motifs-interfacial-interactions-by-co-evolved-analysis-of-d-amino-acid-dehydrogenase-for-stability-enhancement}

\textbf{Hosted file}

\verb`DAADH Figure.docx`  available at \url{https://authorea.com/users/592947/articles/628214-design-of-motifs-interfacial-interactions-by-co-evolved-analysis-of-d-amino-acid-dehydrogenase-for-stability-enhancement}

\textbf{Hosted file}

\verb`DAADH Table.docx`  available at \url{https://authorea.com/users/592947/articles/628214-design-of-motifs-interfacial-interactions-by-co-evolved-analysis-of-d-amino-acid-dehydrogenase-for-stability-enhancement}

\selectlanguage{english}
\FloatBarrier
\end{document}

