An evolution-based designing and characterization of mutants of cyclomaltodextrinase: Molecular modeling and spectroscopic studies

Journal Pre-proof An evolution-based designing and characterization of mutants of cyclomaltodextrinase: Molecular modeling and spectroscopic studies

Jamshid Mehrvand, Nasim Hayati Roodbari, Leila Hassani, Vahab Jafarian, Khosrow Khalifeh PII:

S1386-1425(20)30032-9

DOI:

https://doi.org/10.1016/j.saa.2020.118055

Reference:

SAA 118055

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

31 October 2019

Revised date:

5 January 2020

Accepted date:

9 January 2020

Please cite this article as: J. Mehrvand, N.H. Roodbari, L. Hassani, et al., An evolutionbased designing and characterization of mutants of cyclomaltodextrinase: Molecular modeling and spectroscopic studies, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2020), https://doi.org/10.1016/j.saa.2020.118055

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof An Evolution-Based Designing and Characterization of mutants of cyclomaltodextrinase: Molecular Modeling and Spectroscopic Studies. Jamshid Mehrvanda, Nasim Hayati Roodbaria, Leila Hassanib, Vahab Jafarian*c, Khosrow Khalifeh*c a

Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.

b

c

of

Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, Iran. Department of Biology, Faculty of Sciences, University of Zanjan, Zanjan, Iran.

*

ro

Corresponding authors:

Jo

ur

na

lP

re

-p

Khosrow Khalifeh (Email: [email protected]) and Vahab Jafarian ([email protected]), Department of Biology, Faculty of Sciences, University of Zanjan, Zanjan, Iran.

1

Journal Pre-proof Abstract Cyclomaltodextrinase (CDase) is a member of the alpha-amylase family GH13, the subfamily GH13_20. In addition to CDase and neopullulanase, this subfamily also contains maltogenic amylase. They have common structural features, but different substrate specificity. In current work, a combination of bioinformatics and experimental tools were used for designing and constructions of single and double mutants of a new variant of CDase from Anoxybacillus

of

flavithermus. Considering the evolutionary variable positions 123 and 127 at the dimer interface

ro

of subunits in the alpha-amylase family, these positions in CDase were modified and three

-p

mutants, including A123V, C127Q and A123V/C127Q were constructed. The tertiary structure

re

of WT and mutants were made with the MODELLER program, and the phylogenetic tree of

lP

homologous protein sequences was built with selected programs in Phylip package. Enzyme kinetic studies revealed that the catalytic efficiency of mutants, especially double one, is lower

na

than the WT enzyme. Heat-induced denaturation experiments were monitored by measuring the

ur

UV/Vis signal at 280 nm, and it was found that WT protein is structurally more stable at 25oC. However, it is more susceptible to changes in temperature compared to the double mutant. It was

Jo

concluded that the positions 123 and 127 at the dimeric interface of CDase, not only could affect the conformational stability; but also; the catalytic properties of the enzyme by setting up the active site configuration in the dimeric state. Keywords: Cyclomaltodextrinase, Bioinformatics, Denaturation, Enzyme kinetic, Catalytic efficiency, conformational stability Abbreviations: CDase, cyclomaltodextrinase; GHs, Glycoside hydrolases; A, Alanine; V, Valine; C, Cysteine; Q, Glutamine; WT, wild type; CD, cyclomaltodextrin; IPTG, Isopropyl β-D-1thiogalactopyranoside. 2

Journal Pre-proof 1. Introduction A combination of the primary structure and environmental conditions determine the intrinsic properties of similar enzymes that results in their unique substrate specificity and their ability to operate under various environmental conditions [1,2]. Accordingly, proteins of the same superfamily, gain their unique sequence to overcome the environmental challenges. Finding the mechanisms of adaptation to extreme environmental conditions has commercial advantages in

of

biotechnology and it can be used in protein engineering studies to design enzymes with improved

ro

structural and functional properties [3–5].

-p

Glycoside hydrolases (GHs) are considered as one of the most important groups of enzymes with

re

a wide range of applications in biotechnology [6,7]. They involve in creating and breaking down

lP

complex carbohydrates and glycoconjugates [8]. GHs include several different enzymes with various substrate specificities. Accordingly, a knowledge-based resource dedicated to these

na

enzymes that is known as Carbohydrate-Active enZyme (CAZy database, http://www.cazy.org)

ur

[9]. In the CAZy database, the α-amylase family, also called the GH13 family, contains several

Jo

different subfamilies [10]. Cyclomaltodextrinase (CDase) (EC 3.2.1.54) is a hydrolytic enzyme that catalyzes the biochemical reaction of the conversion of cyclomaltodextrin (CD) into linear maltodextrin [11]. It is a member of the alpha-amylase family GH13, the subfamily GH13_20, the so-called neopullulanase subfamily [12,13]. The subfamily GH13_20 also contains, in addition to cyclomaltodextrinase and neopullulanase, maltogenic amylase [12,14]. CDase has distinct substrate specificity for three types of CDs (α-, β- and γ-CD). It exhibits a 40-86% amino acid sequence identity with maltogenic amylase (EC 3. 2.1.133) and neopullulanase (EC 3.2.1.135) that are responsible for hydrolyzing starch and pullulan, respectively [15,16]. There are over 20 variants of cyclomaltodextrinase, maltogenic amylase, and neopullulanase that share 3

Journal Pre-proof a common sequence ancestor [16]. The crystal structures of these enzymes are available in the protein data bank [16–18]. Structural comparison of the enzymes reveals that they usually possess the N-terminal substrate-binding domain (SBD) and that the dimer formation makes the common active-site cleft between the central (α/β)8-barrel domain and the N-terminal domain [12]. CDase enzymes have isolated and characterized from various sources [19–23]. They are frequently used in those biotechnological processes that involve cyclomaltodextrin metabolism,

of

such as food and pharmaceutical industries [24–27]. Due to their activities under extreme

ro

conditions, thermostable variants of this enzyme produced by thermophilic microorganisms can

-p

offer ideal features for current biotechnological and industrial applications. Recently, a new

re

CDase has identified and characterized from a thermophilic Anoxybacillus flavithermus (GenBank accession number: KT633577.1). It is a thermostable enzyme with optimum

lP

temperature of 65oC that retains its activity after incubation in extreme conditions of temperature

na

[23]. It was shown that α-CD is a more specific substrate for this enzyme. More studies on a mutant of this enzyme have demonstrated that changing the pattern of intra-molecular

ur

electrostatic interaction around catalytic residues can affect the activity of the enzyme [28]. Here,

Jo

two single mutants and their corresponding double mutant for this enzyme were designed and constructed. Designing of mutation were performed considering the occurrence of residues in respective positions at homolog proteins. The structural and functional features of the WT CDase and mutants were investigated using UV/Vis absorption and fluorescence emission spectroscopy. 2. Material and methods 2.1 Materials The

materials

of

the

research

include;

kanamycin

and

IPTG

(Isopropyl

β-D-1-

thiogalactopyranoside) from Fermentas company, nucleotides, and agarose from Invitrogen, 4

Journal Pre-proof nickel-agarose from Qiagen, Expand Long enzyme from Roche, DpnI enzyme from Thermo Fisher Scientific, plasmid extraction and purification kit for PCR products was prepared from Bioneer. Other compounds were purchased from Merck Company. 2.2 Molecular biology pET-28a was used as an expression plasmid for cloning the gene of CDase from Anoxybacillus

of

flavithermus. The enzyme for PCR was the Expand long. The PCR products were purified with

ro

the PCR purification kit, and the final products were mixed with restriction endonuclease DpnI at 37°C for 24 h. The mixture was then transformed into E. coli DH5α cells. Macrogene Company,

-p

Korea performed the sequencing of the mutant gene. Here, two single mutant (A123V and

re

C127Q) and a double mutant (A123V/C127Q) were made. To protein production, a colony of E.

lP

coli (strain BL21) that contains expression plasmid of CDase was grown overnight at 37oC in Luria-Bertani broth (LB) medium. The concentration of kanamycin was 1 µg/ml. 200 µL of this

na

mixture was moved into the same volume of Tryptone Broth (TB) medium and incubated at

ur

37oC until the absorbance at 600 nm of approximately 0.8-0.9 was obtained. After the addition of IPTG into the medium at a final concentration of 0.5 mM, cells were allowed to express the

Jo

protein for 6 h. The culture was then centrifuged at 4000 g for 20 min at 4oC, and the pellet was suspended in Lysis buffer (50 mM Tris, 300 mM NaCl and 10 mM imidazole, pH 8.0). The cells were disrupted by sonication in an ice bath. The solution containing the soluble proteins and other contaminants was then clarified by centrifugation at 12000 g for 20 min at 4oC. Purification of His-tagged fusion proteins was performed by the Nickel-agarose column. The elution buffer (50 mM Tris, 300 mM NaCl and 300 mM imidazole, pH 8.0) was used for separation of given proteins from the column. The purity of purified proteins was evaluated by sodium dodecyl

5

Journal Pre-proof sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) with a 12% (w/v) polyacrylamide gel. The concentration of proteins was determined with the Bradford method [29]. 2.3 Enzyme assay The activity of CDase was measured as described previously [23]. It was determined by measuring the amount of reducing sugar that releases during hydrolysis of 1% soluble CDs in

of

100 mM Hepes buffer (pH 7.0) at 65oC for 20 min. The amount of reducing sugar was measured

ro

using the dinitrosalicylic acid method [30]. One unit of CDase activity under the assay conditions is defined as the amount of the enzyme that releases 1 µmol of reducing sugar per min

-p

[31]. Kinetic parameters were calculated as previously described [28].

lP

re

2.4 Homology modeling

The 3D structure of WT CDase and mutants were constructed by the MODELLER program

na

(Ver. 9.22) [32] using the crystal structure of maltogenic amylase as the template (PDB ID: 1SMA). This structure was selected after running BLAST program by choosing the PDB

ur

database and using the sequence of WT CDase as input for the program. This maltogenic

Jo

amylase has 69.8 similarity with the query sequence. To construct structural models, a pairwise sequence alignment of protein variants with the sequence of template was performed, and the outputs were used for making the tertiary structure of proteins. This process is completed by a two-step energy minimization. The quality of the structural models for each protein variant was assessed by calculation of Z-DOPE score of residues, and the best models were selected via comparing the Z-DOPE ratings. The reliability of modeling was also checked by programs under the SAVE server (https://servicesn.mbi.ucla.edu/SAVES/) [33–36]. 2.5 Sequence Analysis 6

Journal Pre-proof Similarity search of the CDase from Anoxybacillus flavithermus was done with BLAST (Basic Local Alignment Search Tool) program, and some representative proteins with an acceptable level of similarity were selected for sequence analysis. Pairwise global alignment between WT CDase and distinctive protein sequences was done with the Needleman-Wunsch global alignment algorithm under the EMBOSS needle program [37]. Construction of Phylogenetic tree was performed using representative programs in the phylip package [38]. At first, multiple

of

sequence alignment between WT CDase and selected protein sequences was done by the Clustal

ro

Omega program [37], and the output was saved with phylip format. This file was then used as an

-p

input file for the seqboot program to bootstrap and generation of multiple data sets that are

re

resampled versions of the input data set. The output of seqboot was used as input for the Protpars (Protein Sequence Parsimony Method) program. This program allowed any amino acid to change

lP

to any other, and counted the number of such changes needed to evolve the protein sequences on

na

each given phylogeny. The output tree file of the Protpars program was used in the Consensus

ur

tree program, and the final phylogenetic tree was constructed.

Jo

2.6 Fluorescence measurements

The local tertiary structure of the proteins around Trp residues was compared by measuring the intrinsic fluorescence spectra after excitation at 290 nm. Fluorescence measurements were performed at 25°C by a PerkinElmer LS 45 spectrophotometer with a 1 cm path length cuvette. Protein concentration for all fluorescence measurements was 20 μg/mL. Both excitation and emission slits were set to 10 nm, and the spectra were scanned between 310- 410 nm. To evaluate the surface-exposed hydrophobic patches of the WT and mutants; extrinsic fluorescence was measured using 1-Anilino-8-naphthalene sulfonate (ANS) as chromophore; where the concentrations of protein and ANS were 1 and 30 μM, respectively. The emission spectra of 7

Journal Pre-proof ANS-based fluorescence (400-600 nm) were recorded by an excitation wavelength of 350 nm. Both excitation and emission slits were set to 10 nm, and spectra were recorded at a scanning rate of 100 nm/min. 2.5 Thermal denaturation experiments A Jasco UV/Vis spectrophotometer (V-730) equipped with a programmable Peltier was used for

of

conducting the heat-induced denaturation experiments. The temperature scans of protein

ro

solutions were performed by measuring the absorption signal at 280 nm. The protein samples (1 mg/ml) were heated between 25-65°C with the scan rate of 1°C/min and, the signal at 280 nm

-p

was recorded every 2°C. The sigmoid-like thermal denaturation curves were analyzed by fitting

re

the experimental data into the Eq. 1 using Kaliedagraph analysis software [39]:

(1)

ur

na

lP

∆H 1 1 {((αN + βN T) + (αD + βD T) × exp (( R )(T − T))) m y= ∆H 1 1 (1 + exp (( R )(T − T))} m

Jo

Where, αN and βN are the native state baseline and its slope, respectively. αD and βD are denatured state baseline and its slope, respectively, y is the spectroscopic signal, ΔH is the enthalpy change of the denaturation process, Tm is the mid-point of the structural transition, T is the temperature in Kelvin and R is the universal gas constant. Since the change in Gibbs free energy (∆G=∆H-T∆S) in the mid-point of the denaturation curve is zero, therefore, Eqs. 2-4 can be used to calculate the change in entropy of the reactions: ∆H − Tm ∆S = 0

(2)

∆H = Tm ∆S

(3)

8

Journal Pre-proof

∆S =

∆H

(4)

Tm

Using the values of ΔH and ΔS, the change in Gibbs free energy at any temperature (T) can be calculated with Eq. 5: ∆G=∆H-T∆S

(5)

3. Results and discussion

of

3.1 Bioinformatics and description of mutations

ro

Similarity search of the CDase from Anoxybacillus flavithermus in non-redundant protein

-p

sequence databases was performed with the BLAST program, and some representative proteins

re

with an acceptable level of similarity were selected for sequence analysis. Other proteins from GH13_20 subfamily were selected from CAZy database. The complete description of selected

Jo

ur

na

lP

proteins, as well as WT CDase, is provided in Table 1.

9

Journal Pre-proof Table 1. Description of proteins according to their accession Numbers. Cyclomaltodextrinase (CDase) for this study is marked with * sign. The first 7 proteins were selected after running similarity search by the BLAST program using the sequence of CDase (AMB26774) as input. Proteins 9-22 belong to GH13_20 subfamily and were chosen from the CAZy database, http://www.cazy.org. Pairwise Global sequence alignment between WT CDase and distinctive proteins was performed with the EMBOSS needle program.

neopullulanase

Q5BLZ7

CDase maltogenic amylase maltogenic αamylase

C8WUQ7

Jo

P32818

ur

Q9AIV2

Q68KL3

CDase

P29964

cyclodextrinase

Q59226 Q08341

CDase cyclodextrinase maltogenic αamylase maltogenic amylase

Q04977 Q9R9H8

79.9 79.2

69

79.9

68.5

79.5

Geobacillus Anoxybacillus flavithermus AK1 Anoxybacillus tepidamans Anoxybacillus flavithermus Bacillus sp. KSM-1876 Bacillus sp. (in: Bacteria) A2-5A

69.2 98.3 73 83.9 72.6 73

80 99.5 82.8 74.5 59.2 60.6

Bacillus sp. US149

71.5

58.1

Bacillus sp. WPD616

77.2

67.5

76.1

65.7

57.4

43.8

Bacillus thermoalkalophilus ET2

80.4

73.1

Bacillus acidopullulyticus

68.3

56

49.2

38.2

59.8

45.3

66.1 63.8

53.5 48.9

Bacillus licheniformis

45.7

30.8

Bacillus subtilis SUH4-2

63.7

51.8

Geobacillus sp. Gh6

of

Thermus sp. IM6501

Geobacillus stearothermophilus IMA6503 Laceyella sacchari

na

Q52PU5

69 68.2

ro

CDase Neopullulanase maltogenic amylase maltogenic amylase alpha-glycosidase alpha-glycosidase alpha-glycosidase CDase neopullulanase CDase maltogenic αamylase maltogenic αamylase

A7DWA8

100

-p

AKU27292 AKM17981

WP_021322528 WP_003395492 WP_027408977 Q5BLZ6 Q57482 O82982

100

Anoxybacillus flavithermus subsp. flavithermus Geobacillus sp. LC300 Geobacillus sp. 12AMOR1

lP

CDase

ADM86931

Similarity (%)

source

*AMB26774

AAC15072

Identity (%)

Protein Name

re

Accession Number

Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446 Thermoanaerobacter ethanolicus ATCC 33223 / 39E Bacillus sp. (in: Bacteria) Lysinibacillus sphaericus E-244

As shown by black arrows in Fig. 1, CDase contains Ala and Cys in positions 123 and 127; respectively, and these positions in the conservation line of multiple sequence alignment file are 10

Journal Pre-proof occupied with other residues, mainly Val and Gln. Structural comparison at the domain-level with known structures reveals that the loop fragment 120-130 in the current CDase, is the connecting loop of the N-terminal domain and (α/β)8 barrel motif. It is believed that the Nterminal is responsible for dimerization of subunits, and the corresponding region of the Nterminal of one subunit covers a part of the opposite side of the (α/β)8 barrel of the other subunit [17,40]. According to the current evolutionary models, it is believed that variable positions in

of

multiple sequence alignment file of homologous proteins have undergone various types of

ro

mutations during evolutionary time scale. Hence, to better understand the evolutionary

-p

relationships between protein sequences, their alignment file was used for the construction of the

re

phylogenetic tree. Fig. 2 shows the phylogenetic tree of protein sequences made by Phylip package programs. As can be seen from Fig. 2, val and Gln residues are more frequently

lP

occurred at position 123 and 127, respectively. Occurrence of these residues at corresponding

na

positions of variants of all types of Glycoside hydrolases including CDase, alpha-glycosidase, maltogenic amylase and neopullulanase, indicate that these positions in current variant of CDase have

ur

tolerability against mutations. Considering these observations, in this work, new mutants of

respectively.

Jo

CDase were made, where Ala and Cys at positions 123 and 127 were replaced with Val and Gln,

To compare the structural features of protein variants, several 3D structures for WT and mutants (A123V, C127Q and, A123V/C127Q) were made using the MODELLER program, and the best model for individual protein variants was selected based on the Z-Dope score of the structures as well as the ratings of the programs under SAVES server. Fig. 3 shows the Z-Dope score for all residues of the WT and mutants. These structures were then used for further analysis. It is worth to mention that the modeling outputs were also checked by programs under SAVES server, and

11

Journal Pre-proof other structural parameters such as reliability of Ramachandran plots were confirmed. As shown in the upper panel of Fig. 3, each domain contains (β/α)8 barrel structure, which is a typical structural motif in the CD-hydrolyzing enzymes GH13 family [14,41]. The solid surface representation of WT CDase is shown in Fig. 4; where the positions of Ala123 and Cys127 in protein chains are shown in red and green, respectively. From Fig. 4, it can be seen that they are approximately surfaced-exposed residues. Regarding the Hydrophilicity scores of amino acids, it

of

seems that the inclusion of both new residues in this region is thermodynamically favorable [42].

ro

3.2 Spectroscopy

-p

The fluorescence emission was measured to evaluate the changes in the tertiary structure of

re

protein upon mutation. Fig. 5 shows the intrinsic and ANS-based fluorescence spectra of WT and

lP

mutant proteins.

na

Since fluorescence emission upon excitation at 290 nm provides information regarding the microenvironment around Trp residues, it is essential to determine the number and position of

ur

these chromophores in the protein structure. Fig. 6 shows the whole structure of WT CDase;

Jo

where the positions of Trp residues are shown as spheres. It can be seen that each protein chain of the enzyme contains 17 residues of Trp. Therefore, the information of intrinsic fluorescence data of Fig. 5, A, may be averaged over the positions of these chromophores in the whole structure of the protein. It is worth to mention that quenching of Trp fluorescence by proximity to internal quenchers such as His, Asp, and Glu residues leads to the absorption of emitted photons of Trp residues and decreasing the intensity of fluorescence emission. On the other hand, quenching of Trp fluorescence upon exposure to polar solvent molecules, as external quenchers, leads to losing a part of the fluorescence energy and observing the red-shift of the characteristic wavelength of the fluorescence spectrum. 12

Journal Pre-proof According to fluorescence data of Fig. 5, A; no spectral shift in characteristic wavelength of fluorescence emission is observed, demonstrating that the polarity of Trp residues is not changed upon mutation. However, the WT protein has the maximum intrinsic fluorescence intensity compared to other protein variants, indicating that more Trp residues are involved in emission spectra. Therefore, decreasing the fluorescence intensity in mutants, especially in double one, demonstrates that the intrinsic quenchers internally quench emitted photons of some Trp

of

residues. These observations could be interpreted as local structural rearrangements of the

ro

mutants compared with WT protein that results in the proximity of some Trp residues to the

-p

internal quenchers. According to ANS fluorescence data of Fig. 5, B; mutation is accompanied

re

by decreasing the fluorescence intensity. This observation indicates that the number of binding sites for ANS molecules decreases upon mutation. A small red-shift in the ANS fluorescence

ur

3.3 Stability measurements:

na

compared with the WT enzyme.

lP

spectra shows that the binding strength to the ANS molecules is weaker in mutant proteins

Jo

Thermodynamic studies were performed to determine the thermal stability of WT and mutants in a range of temperatures. Fig. 7 shows the heat-induced denaturation curves for protein variants monitored by UV/Vis spectrophotometry. Continuous lines in each panel are the best fitting of experimental data into Eq. 1.

Table 2 provides the numerical values of thermodynamic

parameters of thermal stability measurements.

13

Journal Pre-proof Table 2. Thermodynamic parameters of heat-induced denaturation for WT CDase and its mutants. The values of ΔH and Tm were obtained by modeling the experimental data of thermal denaturation experiments using Eq. 1. The change in entropy of denaturation (ΔS) was calculated by Eq. 4. Thermodynamic parameters Tm (oK) 344.0±0.3

ΔS (cal/mole.K) 828±2

A123V

253±0.6

345.7±0.1

732±2

C127Q

258±0.7

344.2±0.2

749±2

Double

191±0.3

345.0±0.1

of

WT

ΔH (kcal/mol) 285±0.8

554±1

-p

ro

The thermodynamic parameters and their respective standard deviations are reported as the results of three repetitions of the experiments.

re

According to the thermodynamic data of Table 2, the values of Tm are approximately the same for WT and mutants. However, a considerable difference in the enthalpy and entropy changes of

lP

the unfolding reaction between WT and mutants is observed. Denaturation of proteins results in

na

increasing the order of water molecules around non-polar and hydrophobic residues. It also leads to increasing the conformational entropy of the protein chain as a result of breaking the

ur

stabilizing interactions. The net values of ΔH and ΔS of the unfolding reaction is the result of

Jo

these contributions. In other words, the ΔS of hydration is accompanied by decreasing the entropy of bulk water. It acts against denaturation, while ΔS of interactions lead to increasing the conformational entropy of the polypeptide chain and favors denaturation. Similarly, ΔH of hydration favors the denaturation process, and ΔH of interaction is related to the stability of the native state of the protein. Therefore, to consider both parameters together and better understand the thermodynamic stability of protein variants, free energy change of the unfolding reaction (ΔG) at different temperatures were calculated using thermodynamic data of Table 2 into Eq. 5 (Fig. 8). From Fig. 8, it can be seen that the stability of WT protein at 25oC is greater than

14

Journal Pre-proof mutants, and the double mutant has the lowest stability at this temperature. It is also revealed that all protein variants show a linear dependence on temperature with the slops 0.82±0.09, 0.74±0.07, 0.72±0.08 and 0.54±0.07 kcal/mole/C for WT, C127Q, A123V, and double mutants, respectively. Hence, the gradient of the stability change for double mutant as a function of temperature is slower than other protein variants, and that of WT protein is the greatest. This observation shows that WT protein is more susceptible to changes in temperature; while, the

of

double mutant is less sensitive to raise temperature compared with other protein variants. By this

ro

modeling, it is predicted that ∆G of the unfolding reactions for all proteins converges to the same

-p

value at higher temperatures near 70oC.

re

3.4 kinetic parameters

na

lP

The kinetic parameters of protein variants are shown in Table 3, demonstrating that the WT enzyme has improved kinetic parameters compared with mutants. As can be seen from Table 3, catalytic efficiency for all forms of substrates, including α-, β, and γ-CD decreases upon mutation.

α

β

γ

Enzyme WT A123V C127Q Double WT A123V C127Q Double WT A123V C127Q Double

Jo

Substrate

ur

Table 3. Kinetic parameters for WT CMDase and mutants at 25oC. Data for WT protein were taken from [28]. K m (mg/ml) 3.99±0.3 4.35±0.2 4.12±0.7 4.71±0.4 3.45±0.5 4.07±0.1 3.81±0.7 4.30±0.5 2.98±0.4 3.65±0.5 3.19±0.1 3.90±0.7

15

K cat (s−1 ) 580±0.9 410±0.3 498±0.7 373±0.6 382±0.5 329±0.5 349±0.4 305±0.8 308±0.8 254±0.5 291±0.4 231±0.7

K cat /K m (mg/ml. s) 145±11 94±0.4 120±3 79±4 111±16 80±3 91±4 70±5 103±14 69±3 91±4 59±4

Journal Pre-proof Generally, the GH13 family is multidomain proteins; each has an N-terminal region composed of several β-strands. It is connected to the catalytic region that has (β/α)8-barrel shape, and extended to a Greek key motif at C-terminal [15,43,44]. Accordingly, it appears that the mutations at the dimer interface lead to changing the active site configuration and catalytic properties of these enzymes in the dimeric state [17]. Additionally, data of Table 3 would be explained in the statistical thermodynamics paradigm, concerning the proposed model of Fig. 8, obtained from

of

heat-induced denaturation experiments. According to this model, the more positive value of

ro

Gibbs free energy change at 25oC for WT protein is a consequence of fractional amounts of

-p

equilibrium constant, as the ratio of the population of the unfolded to folded state at equilibrium

re

condition. Therefore, according to the familiar thermodynamic equation (∆G=-RTlnK), it may be concluded that the effective concentration of enzyme molecules at the assay condition is more

lP

significant for WT enzyme compared with mutants.

na

3.5 Conclusions

ur

Regarding the general tertiary structure of GH13 family, concerning the presence of super

Jo

secondary structures such as TIM-barrel and Greek key motifs, the loops have critical structural roles and contribute to the overall architecture of protein via connecting successive secondary structural elements. The variability in the sequence of corresponding loops at similar positions is one of the primary sources of the differences in the sequence of the α-amylase family. The loop 120-130 between the first and second motifs is involved in the dimerization of subunits. It can affect the active site configuration of the catalytic site as well as the stability of the folded state of the dimeric form of the enzyme. Observation of variable amino acids in conservation lines upon multiple sequence alignment between similar enzymes demonstrates that this loop has high tolerability against mutation that could offer various structural and functional features to the 16

Journal Pre-proof respective variant of the enzyme. However, further studies are needed to elucidate the exact role of this loop in GH13 family. Acknowledgments Financial support for this work was provided by the Science and Research Branch of the Islamic Azad University of Tehran. We appreciate the university of Zanjan , University of Tehran and

of

Institute for Advanced Studies in Basic Sciences of Zanjan for technical support of the research.

ro

The authors have declared no conflict of interest.

O.P. Ward, M. Moo-Young, Thermostable enzymes, Biotechnol. Adv. 6 (1988) 39–69.

[2]

Peter K. Robinson, Enzymes: principles and biotechnological applications, Essays

na

Biochem. 59 (2015) 1–41.

P. Fernandes, Enzymes in food processing: A condensed overview on strategies for better

ur

[3]

lP

doi:10.1016/0734-9750(88)90573-3.

re

[1]

-p

References:

[4]

Jo

biocatalysts, Enzyme Res. (2010) 1–19. doi:10.4061/2010/862537. S.M. Basheer, S. Chellappan, Enzyme engineering, in: Bioresour. Bioprocess Biotechnol., 2017: pp. 151–158. doi:10.1007/978-981-10-4284-3_6. [5]

J.M. Woodley, New opportunities for biocatalysis: making pharmaceutical processes greener, Trends Biotechnol. 26 (2008) 321–327. doi:10.1016/j.tibtech.2008.03.004.

[6]

A.S. Melim Miguel, T. Souza, E.V. da Costa Figueiredo, B.W. Paulo Lobo, G. Maria, Enzymes in Bakery: Current and Future Trends, in: Food Ind., 2013. doi:10.5772/53168.

[7]

S.E. Jujjavarapu, S. Dhagat, Evolutionary Trends in Industrial Production of α-amylase, 17

Journal Pre-proof Recent Pat. Biotechnol. 13 (2018) 4–18. doi:10.2174/2211550107666180816093436. [8]

S. JaneCek, B. Svensson, E.A. MacGregor, α-Amylase: An enzyme specificity found in various families of glycoside hydrolases, Cell. Mol. Life Sci. 71 (2014) 1149–1170. doi:10.1007/s00018-013-1388-z.

[9]

B.I. Cantarel, P.M. Coutinho, C. Rancurel, T. Bernard, V. Lombard, B. Henrissat, The

of

Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics,

ro

Nucleic Acids Res. (2009) 233–238. doi:10.1093/nar/gkn663.

-p

[10] J. Zhou, Z. Li, H. Zhang, J. Wu, X. Ye, W. Dong, M. Jiang, Y. Huang, Z. Cui, Novel maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the conversion

re

of maltooligosaccharides and soluble starch to maltose, Appl. Environ. Microbiol. 84

lP

(2018) 1–15. doi:10.1128/AEM.00152-18.

na

[11] J.A. De Pinto, L. Leon Campbell, Purification and Properties of the Cyclodextrinase of

ur

Bacillus macerans, Biochemistry. 7 (1968) 121–125. doi:10.1021/bi00841a016. [12] A. Kuchtová, Š. Janeček, Domain evolution in enzymes of the neopullulanase subfamily,

Jo

Microbiol. (United Kingdom). 162 (2016) 2099–2115. doi:10.1099/mic.0.000390. [13] L. Plaza-Vinuesa, O. Hernandez-Hernandez, F.J. Moreno, B. De Las Rivas, R. Munõz, Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1, Microb. Cell Fact. 18 (2019) 1–11. doi:10.1186/s12934-019-1237-3. [14] M.R. Stam, E.G.J. Danchin, C. Rancurel, P.M. Coutinho, B. Henrissat, Dividing the large glycoside hydrolase family 13 into subfamilies: Towards improved functional annotations of α-amylase-related proteins, Protein Eng. Des. Sel. 19 (2006) 555–562.

18

Journal Pre-proof doi:10.1093/protein/gzl044. [15] K.H. Park, T.J. Kim, T.K. Cheong, J.W. Kim, B.H. Oh, B. Svensson, Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family, Biochim. Biophys. Acta - Protein Struct. Mol. Enzymol. 1478 (2000) 165–185. doi:10.1016/S0167-4838(00)00041-8.

of

[16] H.S. Lee, M.S. Kim, H.S. Cho, J.I. Kim, T.J. Kim, J.H. Choi, C. Park, H.S. Lee, B.H. Oh,

ro

K.H. Park, Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other, J. Biol. Chem. 14 (2002) 21891–21897.

-p

doi:10.1074/jbc.M201623200.

re

[17] J.S. Kim, S.S. Cha, H.J. Kim, T.J. Kim, N.C. Ha, S.T. Oh, H.S. Cho, M.J. Cho, M.J. Kim,

lP

H.S. Lee, J.W. Kim, K.Y. Choi, K.H. Park, B.H. Oh, Crystal structure of a maltogenic

na

amylase provides insights into a catalytic versatility, J. Biol. Chem. 274 (1999) 26279– 26286. doi:10.1074/jbc.274.37.26279.

ur

[18] H. Hondoh, T. Kuriki, Y. Matsuura, Three-dimensional structure and substrate binding of

Jo

Bacillus stearothermophilus neopullulanase, J. Mol. Biol. 326 (2003) 177–188. doi:10.1016/S0022-2836(02)01402-X. [19] S. Kitahata, M. Taniguchi, S.D. Beltran, T. Sugimoto, S. Okada, S. Duque Beltran, Agricultural and Biological Chemistry Purification and Some Properties of Cyclodextrinase from Bacillus coagulans Purification and Some Properties of Cyclodextrinase from Bacillus coagulans, Agric. Biol. Chem. Agric. BioI. Chern. 47 (1983) 1441–1447. doi:10.1080/00021369.1983.10865801. [20] S.M. Podkovyrov, J.G. Zeikus, Structure of the gene encoding cyclomaltodextrinase from 19

Journal Pre-proof Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli, J. Bacteriol. 174 (1992) 5400–5405. doi:10.1128/jb.174.16.54005405.1992. [21] T. Oguma, M. Kikuchi, K. Mizusawa, Purification and some properties of cyclodextrinhydrolyzing enzyme from Bacillus sphaericus, BBA - Gen. Subj. 1036 (1990) 1–5.

of

doi:10.1016/0304-4165(90)90205-B.

ro

[22] H. Bender, Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp., Appl. Microbiol. Biotechnol. 39 (1993) 714–719.

-p

doi:10.1007/BF00164455.

re

[23] N. Aliakbari, Z. Mirzaee, V. Jafarian, K. Khalifeh, M. Salehi, Genetic and biochemical

lP

characterization of a novel thermostable cyclomaltodextrinase from Anoxybacillus

na

flavithermus, Starch - Stärke. (2018) 1–11. doi:10.1002/star.201800133. [24] A. El-Fallal, M. Abou, A. El-Sayed, N. Omar, Starch and Microbial α-Amylases: From

ur

Concepts to Biotechnological Applications, in: C.-F. Chang (Ed.), Carbohydrates -

Jo

Compr. Stud. Glycobiol. Glycotechnol., IntechOpen, 2012: pp. 460–488. doi:10.5772/51571.

[25] T. Loftsson, D. Duchêne, Cyclodextrins and their pharmaceutical applications, Int. J. Pharm. 329 (2007) 1–11. doi:10.1016/j.ijpharm.2006.10.044. [26] M. Singh, R. Sharma, U.C. Banerjee, Biotechnological applications of cyclodextrins, Biotechnol. Adv. 20 (2002) 341–359. doi:10.1016/S0734-9750(02)00020-4. [27] E. Fenyvesi, M. Vikmon, & L. Szente, Cyclodextrins in Food Technology and Human

20

Journal Pre-proof Nutrition: Benefits and Limitations, Crit. Rev. Food Sci. Nutr. 56 (2016) 1981–2004. doi:10.1080/10408398.2013.809513. [28] Z. Mirzaee, V. Jafarian, S.A. Shirdel, K. Khalifeh, Structural and functional consequences of replacement of His403 with Arg near the catalytic site of Anoxybacillus flavithermus cyclomaltodextrinase, Enzyme Microb. Technol. 131 (2019) 109421–109427.

of

doi:10.1016/j.enzmictec.2019.109421.

ro

[29] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities

254. doi:10.1016/0003-2697(76)90527-3.

-p

of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–

re

[30] G.L. Miller, Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar,

lP

Anal. Chem. 31 (1959) 426–428. doi:10.1021/ac60147a030.

ur

6879(55)01021-5.

na

[31] P. Bernfeld, Amylases, α and β, Methods Enzymol. 1 (1955) 149–158. doi:10.1016/0076-

[32] A. Fiser, A. Šali, MODELLER: Generation and Refinement of Homology-Based Protein

Jo

Structure Models, Methods Enzymol. 374 (2003) 461–491. doi:10.1016/S00766879(03)74020-8.

[33] J.U. Bowie, R. Luthy, D. Eisenberg, A Method to Identify Protein Sequences That Fold into a Known 3-Dimensional Structure, Science (80-. ). 253 (1991) 164–170. doi:DOI 10.1126/science.1853201. [34] C. Colovos, T.O. Yeates, Verification of protein structures: Patterns of nonbonded atomic interactions, Protein Sci. 2 (1993) 1511–1519. doi:10.1002/pro.5560020916.

21

Journal Pre-proof [35] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the stereochemical quality of protein structures, J. Appl. Crystallogr. 26 (1993) 283–291. doi:10.1107/S0021889892009944. [36] J. Pontius, J. Richelle, S.J. Wodak, Deviations from Standard Atomic Volumes As A Quality Measure for Protein Crystal Structures, J. Mol. Biol. 264 (1996) 121–36.

of

doi:10.1006/jmbi.1996.0628.

ro

[37] F. Madeira, Y. mi Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, A.R.N. Tivey, S.C. Potter, R.D. Finn, R. Lopez, The EMBL-EBI search and sequence analysis

-p

tools APIs in 2019, Nucleic Acids Res. 2 (2019) W636–W641. doi:10.1093/nar/gkz268.

re

[38] J. Felsenstein, PHYLIP - Phylogeny Inference Package (Version 3.2), Cladistics. (1989)

lP

164–166.

na

[39] S.A. Shirdel, K. Khalifeh, Thermodynamics of protein folding: methodology, data analysis and interpretation of data, Eur. Biophys. J. 48 (2019) 305–316.

ur

doi:10.1007/s00249-019-01362-7.

Jo

[40] S. Kamitori, S. Kondo, K. Okuyama, T. Yokota, Y. Shimura, T. Tonozuka, Y. Sakano, Crystal structure of thermoactinomyces vulgaris R-47 α-Amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 Å resolution, J. Mol. Biol. 287 (1999) 907–921. doi:10.1006/jmbi.1999.2647. [41] K. Unban, A. Kanpiengjai, S. Lumyong, T.H. Nguyen, D. Haltrich, C. Khanongnuch, Molecular structure of cyclomaltodextrinase derived from amylolytic lactic acid bacterium Enterococcus faecium K-1 and properties of recombinant enzymes expressed in Escherichia coli and Lactobacillus plantarum, Int. J. Biol. Macromol. 107 (2018) 898– 22

Journal Pre-proof 905. doi:10.1016/j.ijbiomac.2017.09.060. [42] J. Kyte, R.F. Doolittle, A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157 (1982) 105–132. doi:10.1016/0022-2836(82)90515-0. [43] E.A. MacGregor, S. JaneCek, B. Svensson, Relationship of sequence and structure to specificity in the α-amylase family of enzymes, Biochim. Biophys. Acta - Protein Struct.

of

Mol. Enzymol. 1546 (2001) 1–20. doi:10.1016/S0167-4838(00)00302-2.

ro

[44] S. Janecek, B. Svensson, B. Henrissat, Domain evolution in the α-amylase family, J. Mol.

-p

Evol. 45 (1997) 322–331. doi:10.1007/PL00006236.

re

Figure Legends

na

lP

Figure 1. Multiple sequence alignment of template sequences to CDase (AMB26774). The sequences were aligned by the CLUSTALW program, and the alignment file was depicted by the ESPript server. Residue numbers are labeled according to the WT-AMB26774 sequence (CDase). The complete conserved residues in the conservation line are shown in white color and are shaded in red. Black arrows indicate the positions of mutation

ur

Figure 2. Phylogenetic tree of the protein sequences. The tree was constructed by a sequence of programs in the Phylip package. The red-colored right-hand side includes residues at positions 123 and 127 of the corresponding protein, which is shown before and after / sign, respectively.

Jo

Figure 3. Homology modeling and structural analysis. Upper panel: superimposed structures of WT CDase and mutants. The structure is constructed in the dimeric form containing subunits A and B. WT protein, A123V, C127Q, and double mutants are shown by green, magenta, yellow and blue, respectively. Superposition was performed by the Chimera program. Lower panel: The quality of selected structural models. Z-DOPE score of residues was calculated by running the MODELLER program using appropriate command lines for evaluating the structures. Negative values of Z-DOPE scores indicate that the structures have sufficient quality for further analysis. The quality of structures was also checked by programs under the online SAVES server. Figure 4. The solid surface shape of WT CDase. The Ala123 and Cys127 are shown with red and green colors, respectively. The tertiary structure of the protein was made by the MODELLER, and the graphical representation was done by the Chimera program. Figure 5. Fluorescence spectra data of WT and mutant proteins. Intrinsic fluorescence was measured with the excitation wavelength at 290 nm, and the emission spectra were recorded between 310-410 nm. Extrinsic fluorescence was measured using ANS as extrinsic

23

Journal Pre-proof chromophores with the excitation wavelength of 350 nm, and the emission spectra were recorded between 400-600 nm. Figure 6. The position of Trp residues in the structure of WT CDase. Each chain of CDase has 17 residues of Trp that are shown as spheres. The structural model of protein was made by the MODELLER program, and the graphical representation of the protein was done by Chimera software.

of

Figure 7. Heat-induced denaturation of protein variants. The unfolding process as a function of temperature was monitored by UV/Vis spectroscopy at 280 nm. Separate points are experimental measurements of absorbance, and continuous lines are the best fitting of data into Eq. 1. Modeling of the experimental data was performed by Kaliedagraph fitting software that results in obtaining numerical values of the enthalpy change (ΔH) and mid-point of the structural transition (Tm).

Jo

ur

na

lP

re

-p

ro

Figure 8. Temperature dependence of Free energy change of the unfolding reaction. The values of ΔG for individual temperatures were calculated by Eq. 5 and thermodynamics data of Table 1.

24

Journal Pre-proof Credit author statement: J. Mehrvand: Developing of research conception, molecular Biology, Enzyme assay and data collection, Bioinformatics, data analysis N. Hayati Roodbari: Developing of research conception, Bioinformatics L. Hassani: Developing of research conception, Spectroscopy V. Jafarian: Developing of research conception, molecular Biology and data collection, Data analysis

Jo

ur

na

lP

re

-p

ro

of

K. Khalifeh: Developing of research conception, Bioinformatics, Data Analysis, WritingReviewing and Editing of the manuscript.

25

Journal Pre-proof Declaration of competing interest

Jo

ur

na

lP

re

-p

ro

of

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.

26

Journal Pre-proof Highlights   

Positions 123 and 127 in cyclomaltodextrinase (CDase) were changed the sequence conditions of other Glycoside hydrolases. Thermal stability of WT protein at 25oC is greater than mutants. WT protein is more sensitive to changes in temperature,



The loop in the dimeric interface has critical role in catalytic efficiency and

Jo

ur

na

lP

re

-p

ro

of

conformational stability.

27

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8