Improvement of catalytic efficiency and thermal stability of l -asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus

Accepted Manuscript Title: Improvement of catalytic efficiency and thermal stability of L-Asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus Authors: Yue Feng, Song Liu, Cuiping Pang, Hui Gao, Miao Wang, Guocheng Du PII: DOI: Reference:

S1359-5113(18)31237-6 https://doi.org/10.1016/j.procbio.2019.01.001 PRBI 11546

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

12 August 2018 13 December 2018 3 January 2019

Please cite this article as: Feng Y, Liu S, Pang C, Gao H, Wang M, Du G, Improvement of catalytic efficiency and thermal stability of L-Asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus, Process Biochemistry (2019), https://doi.org/10.1016/j.procbio.2019.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Improvement of catalytic efficiency and thermal stability of L-Asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus

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Yue Feng1, 2, Song Liu2, 3#, Cuiping Pang2, 3, Hui Gao2, 3, Miao Wang4, Guocheng Du3, 5

School of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 311402, China

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National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China

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School of Biotechnology, Jiangnan University, Wuxi 214122, China

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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

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The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University,

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Corresponding authors: Song Liu, Tel.: +86-510-85918307, Fax: +86-510-85918309, E-mail:

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Wuxi 214122, China

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[email protected];

Graphical abstract

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20-29) N-terminal N-terminus flexible loop flexible (residues loop

Tyr30

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Thr16 ligand ASP

Bacillus subtills L-asparaginase (ASN)

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Reduce the flexibility of the N-terminal flexible loop

The flexibility of residues20-29 in ASN mutants

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Specific activity and thermal stability of ASN mutants

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Highlights 

Enhanced catalytic activity of L-asparaginase by modifying highly flexible loop.

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Reducing the flexibility of N-terminal loop contributes to L-asparaginase catalysis.

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A mutant with enhanced specific activity and thermal stability was obtained.

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High specific activity will reduce the dosage and usage cost of L-asparaginase.

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ABSTRACT

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Herein, the catalytic efficiency and thermal stability of L-Asparaginase (ASN, EC 3.5.1.1) from Bacillus subtilis 168

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were enhanced by modifying its N-terminus highly flexible loop. A continuous non-conserved region (residues20-29)

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within the N-terminal loop of the ASN was identified and substituted with the structurally equivalent region of

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ASNs from Escherichia coli, Helicobacter pylori, Wolinella succinogenes, Pseudomonas aeruginosa, Erwinia

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chrysanthemi, and Pectobacterium carotovorum, yielding the ASN loop variants L1, L2, L3, L4, L5, and L6, respectively. In contrast to the wild-type enzyme, L6 exhibited the highest increase (2.1-fold) in specific activity.

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Molecular dynamics analysis suggested a negative correlation between the flexibility of the continuous non-conserved region and specific activity. Saturation mutagenesis and complex mutation on flexible residues Ala26

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and Gly29 of L6 obtained a double variant L6-A26N/G29F with 3.44-fold and 7.76-fold increases in specific activity and half-life at 65°C in comparison with wild-type enzyme, respectively. As suggested by structural analysis,

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the reduced flexibility of the continuous non-conserved region benefit the correct orientation of the active site Thr16 and closure of the substrate pocket. Our results demonstrated that reducing the flexibility of the highly flexible loop was an efficient method to enhance the catalytic efficiency and thermal stability of ASN.

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Keywords: Bacillus subtilis; L-Asparaginase; flexible loop; residue modification; catalytic efficiency; thermal

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stability

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INTRODUCTION L-Asparaginase (ASN, EC 3.5.1.1) catalyses the conversion of L-asparagine into L-aspartic acid and ammonia (1). Currently, two kinds of microorganisms ASN (Type I and II) have been identified based on their substrate affinity,

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quaternary structure and localization (2). Owing to a higher affinity for substrate asparagine, type II ASN has been widely applied in the production of acrylamide-free food and in treating acute lymphoblastic leukaemia (3).

It has been reported that the type II ASNs from different resources showed different catalytic efficiencies and

specific activities (4-9). High catalytic efficiency and specific activity of ASN are desirable characteristics for its

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effective application. Therefore, identifying and modifying key residues that affect the catalysis of ASN will benefit

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its uses in food processing and medical treatment.

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Recently, a variety of residues adjacent to the catalytic cavity of ASN have been modified to improve its catalytic efficiency. For example, Long et al. (10) reported that mutation of residues adjacent to the active site of

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Bacillus subtilis ASN affected its specific activity and catalytic efficiency. Similarly, the catalytic efficiency of

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hyperthermophilic ASN was enhanced by 1.61-fold through modifying the residues adjacent to the catalytic cavity(11). In addition, loops constituting the active cavity were important for protein function, stability, and catalysis (12). Modification of the highly flexible loops surrounding the active cavity is now a well-established and

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efficient strategy for improving specific activity and catalytic efficiency (13).

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The crystal structures of type II ASNs from several microorganisms have been resolved, including E. coli (14),

Wolinella succinogenes (15), Erwinia chrysanthemi (16), Erwinia carotovora (17), and Helicobacter pylori (18). As suggested by these crystal structures, a highly flexible loop was located at the N-terminus of type II ASNs, such as residues12-25 in E. coli ASN (EcA) (14) and residues 1632 in E. chrysanthemi ASN (ErA) (16). Two active residues (Thr12 and Tyr25 in EcA) were found to be strictly conserved in the highly flexible loop of the ASNs from different 5

resources (19). It was shown that the N-terminal highly flexible loop of the ErA exhibited closed and open conformations parallel to the active and inactive states, respectively (20). The closed conformation in which the catalytic Thr15 was positioned into its active conformation was induced by substrate binding, and the rate of the

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closure affected the catalytic activity (20). Based on crystal structures of W. succinogenes ASN (WoA) and its

variants with substrates, Nguyen et al. (19) demonstrated the existence of a conserved three-hinge model of the

N-terminal flexible loop that navigated between its closed and open conformations among bacterial ASNs. These findings suggested that the catalytic activity of the ASN could be enhanced by modifying its N-terminal highly

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flexible loop.

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In our previous report, a type II ASN (ansZ) (21, 22) from B. subtilis 168 was successfully expressed in B.

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subtilis WB600 with a yield of 2.5 g/L ASN protein (23), demonstrating the highest yield of type II ASN by microbial fermentation. In this study, a continuous non-conserved region, residues20-29 (ADQSKTSTTEY) in the

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N-terminal flexible loop of B. subtilis ASN (Fig. 1), was identified to be responsible for its catalytic activity. Loop

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replacement, single-site saturation mutagenesis and complex mutation at this continuous non-conserved region were conducted to improve the ASN specific activity and catalytic efficiency. The mechanisms underlying the enhanced

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catalytic activity were investigated.

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Materials and Methods

Strains and plasmids B. subtilis WB600 (The Bacillus Genetic Stock Center, Columbus, OH, USA) was the host for ASN expression. E. coli JM109 (Novagen, Madison, WI, USA) was used for plasmid construction, and plasmid pP43NMK/ASNΔ25/B2 6

used for B. subtilis ASN expression was constructed in our previous study (23).

Structural analysis

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SWISS-MODEL server (http://swissmodel.expasy.org/) was used to model the structures of B. subtilis 168 ASN and its variants using the crystal structure of ErA (PDB code 1HG0, 1.8 Å resolution) as a template (16). The

combinatorial extension method (24) was also used to calculate the root mean square deviation (RMSD, 0.1 Å)

between the -carbon backbone of the template and modelled structures. According to the combinatorial extension

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process, the sequence identity similarity between B. subtilis ASN and the template was 59.1%. The

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three-dimensional structure of ligand L-aspartic acid was copied from that of EcA- L-aspartic acid complex (PDB

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code 2him, 1.82 Å resolution) (25).

To optimise modelling structure, molecular dynamic (MD) simulations were conducted using NAMD

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implementing the CHARMM force field (http://www.ks.uiuc.edu/Research/namd/). The structures were immersed in

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a rectangular box containing water molecules, and the distance between any protein atom and the edge of the box was fixed to 15 Å. Following the addition of Na+ (0.15 M) to balance the negative charges, the system was minimised using the steepest descent method. After MD simulations conducted at 300 K, plots of RMSD of protein

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backbone were calculated for 20 ns, and all structures reached equilibrium after 12 ns of simulation (data not shown).

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Root Mean Square Fluctuation (RMSF) values that reflect the fluctuation of individual residues at 300 K was also calculated for residues536 which contains the N terminal flexible loop. The post assays were conducted using Visual Molecular Dynamics (http://www.ks.uiuc.edu/Research/vmd/). The distances between the active site Thr16 and Tyr30 were calculated after all structures reached equilibrium. PyMOL (http://www.pymol.org) or Discovery Studio 4.0 (BIOVIA, San Diego, CA, USA) were used to produce the graphical molecular representations. 7

Construction of plasmids encoding ASN with replaced residues20-29 Sequence substitutions of residues20-29 in B. subtilis 168 ASN were performed through whole-plasmid polymerase chain reaction (PCR) using a specific forward primer and a constant reverse primer (R1) (Table 1). For replacing the

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residues20-29 of B. subtilis ASN with the structurally equivalent residues from EcA (14), H. pylori ASN (HpA) (18), WoA (15), Pseudomonas aeruginosa ASN (PgA) (6), ErA (16), and Pectobacterium carotovorum ASN (PcA) (8), F1, F2, F3, F4, F5, and F6 were used as the forward primer, respectively (Table 1). After mixing the PrimeSTAR

polymerase (Takara, Dalian, China), template (plasmid pP43NMK/ASNΔ25/B2) and the variant primers, PCR was

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conducted using the followed amplification program: an initial 3 min of denaturation at 98°C prior to 34 cycles

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including 15 s at 98°C, 10 s at 60°C, and 7 min at 72°C. To eliminate the primary template, Dpn I was used to digest

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the PCR reaction mixtures. Then, these PCR products were purified and transformed into E. coli JM109, yielding the

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plasmids expressing the B. subtilis ASN with residues20-29 from EcA, HpA, WoA, PgA, ErA, and PcA, respectively.

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Construction of plasmids encoding ASN with saturation mutagenesis Saturation mutagenesis was conducted at Ala26 and Gly29 of the B. subtilis ASN with residues20-29 from PcA through whole-plasmid PCR using degenerate primers (Table 1). The degenerate primers containing NNK (forward

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primer) and MNN (reverse primer) at Ala26 and Gly29 of the B. subtilis ASN with residues20-29 from PcA were used

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for saturation mutagenesis at the corresponding mutation sites, where N represents the bases T, A, C, or G, K contains T and G, and M symbolizes A or C (Table 1). PCR was conducted using the followed amplification program: an initial 3 min of denaturation at 98°C prior to 34 cycles including 15 s at 98°C, 10 s at 60°C, and 7 min at 72°C. To eliminate the primary template, Dpn I was used to digest the PCR reaction mixtures. Then, these PCR reactions were purified and transformed into B. subtilis WB600 for ASN expression. Based on these preliminary results of 8

saturation mutagenesis, double mutation was generated through combining the two positive variants using the primer A26N/G29F listed in Table 1.

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Expression of ASN

For flask cultivation, each recombinant B. subtilis strain was cultured in 25 mL LB medium supplemented with

kanamycin (50 μg/mL) overnight at 37°C. Then, 4 % (v/v) of overnight culture in LB medium was transferred into

was collected by centrifuging the samples at 12, 000 rpm for 5 min.

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fermentation medium (23) containing antibiotics and cultured for 48 h. To determine ASN activity, the supernatant

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To screen variants for enhanced activity, each recombinant B. subtilis strain was inoculated into 800 μL

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fermentation medium (23) supplemented with kanamycin (50 μg/mL) in 96-well deep plates and cultured at 37°C with 220 rpm for 48 h. Two hundred colonies were screened for each saturation mutagenesis sites. The recombinant

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B. subtilis strain with the ASN activity higher than wild-type enzyme was identified as the strain producing ASN

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with enhanced catalytic activity.

Measurement of ASN activity

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ASN activity was determined by measuring the release of ammonia as described by Deokar et al (26) with

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L-asparagine as the substrate. After enzymatic reaction, the quantity of ammonia was calculated using Nessler’s reagent (Xiya reagent, Shandong, China) by measuring the absorbance at 436 nm. One unit of ASN activity was defined as the amount of enzyme liberating 1 μmol of NH 3 per min at 37ºC. Specific activity was defined as the number of units of activity per mg of protein.

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Purification of ASN As the wild-type ASN was previously constructed with an N-terminal 6 x His tag (23), all of the variants were generated as His-tag fusions in this study. The purification of recombinant ASNs were performed with nickel affinity

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column (HisTrapTM FF crude; GE Healthcare, Salt Lake City, UT, USA). Before sample loading, the nickel column was equilibrated with binding buffer (20 mM K2HPO4-KH2PO4, pH 7.5). Elution buffer (20 mM K2HPO4-KH2PO4, 500 mM imidazole, pH 7.5) was used to elute recombinant ASNs with a gradient elution program. The purified

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ASNs were stored at 4°C until further analysis.

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Characterization of ASN

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Apparent kinetic parameters (Km and kcat) were as described in our previous study (23). Apparent kinetic parameters (Km and kcat) were determined in 20 mM K2HPO4-KH2PO4 buffer containing 1.89 to 18.9 mM L-asparagine as the

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substrate at 37°C. Data were fitted by Lineweaver–Burk plot and used to count the kinetic parameters Vmax and Km.

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The kcat was calculated by the equation kcat = Vmax/[E], in which [E] is the concentration of ASN (40 U/mL). Thermal stability was assayed as described in our previous study (23). The optimum temperature of ASN was tested between 30°C and 80°C at pH 7.5. The half-life at 65°C was used to indicate the thermal stability, which

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coincided with a first-order curve. The value of half-life (t1/2) of ASN at 65°C was calculated by linear fitting of ln

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(residual activity) versus the incubation time. Nano-differential scanning calorimeter (TA Instruments, New Castle, DE, USA) was used to determine the

melting temperature (Tm) of ASNs. Briefly, protein samples were dissolved to 250 μg/mL in 20 mM K2HPO4-KH2PO4 buffer (pH 7.5), which was degassed under vacuum prior to measurements. K2HPO4-KH2PO4 buffer was used as a control. After an initial 10 min of equilibration at 20°C, protein unfolding data was monitored 10

between 20°C and 100°C at a scan rate of 2.0°C/min. Scans were calculated using the Origin software package

Sodium dodecyl sulphate polyacrylamide gel electrophoresis assay

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(OriginLabs, Northampton, MA, USA).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted on a NuPAGETM 10%

Bis-Tris Gel with MOPS running buffer (Invitrogen, Carlsbad, CA, USA), and Coomassie Brilliant Blue G250 was used to stain proteins. Each sample for SDS-PAGE was prepared according to the protocol provided by Invitrogen.

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In brief, 30 μL culture supernatant of each recombinant strain was mixed with 10 μL NuPAGE LDS Sample Buffer

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(4X); the mixture was heated at 70˚C for 10 minutes and then ready for SDS-PAGE analysis. The purification

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quality of enzyme variants was detected by SDS-PAGE assay. Protein concentration was measured using the

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Spectrum analysis

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Bradford method (27) with bovine serum albumin as a standard.

Circular dichroism and fluorescence spectroscopy analysis were performed as described in our previous study (13).

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RESULTS

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Structural modelling and sequence alignment of B. subtilis ASN The crystal structure of ASN from E. chrysanthemi (PDB code 1HG0), which shares 59.1% sequence identity with the enzyme from B. subtilis 168, has been determined (16). Based on this crystal structure, we modelled the three-dimension structure of B. subtilis ASN which was composed of four homo-subunits (Fig. 2). According to the previous study (16), the residues14-36 (GGTIAGADQSKTSTTEYKAGVVG) of B. subtilis ASN formed the 11

N-terminal highly flexible loop containing the two active site residues Thr16 and Tyr30, and the flexible loop acting as a lid that covers the catalytic cavity and subsequently assists substrate binding and catalysis (16). Sequence alignment of the B. subtilis ASN and six other ASNs (EcA, HpA, WoA, PgA, ErA and PcA) was performed using

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ClustalX2 (http://www.ebi.ac.uk/Tools/msa/clustalo/). It was showed that the residues20-29 (ADQSKTSTTE) of B. subtilis ASN constituted a continuous non-conserved region (Fig. 1). Moreover, residues20-29 were located on the

subunit interface which was critical for the catalytic reaction (Fig. 2). Thus, the residues20-29 of B. subtilis ASN were

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Sequence substitution at the N-terminal loop of B. subtilis ASN

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selected as the modification sites.

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To improve the catalytic activity, the residues20-29 of B. subtilis ASN were replaced with the structurally equivalent residues of EcA, HpA, WoA, PgA, ErsA and PcA (Fig 1), yielding B. subtilis ASNs with replaced N-terminal loop

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L1, L2, L3, L4, L5, and L6, respectively. As the corresponding expression plasmids were constructed based on the

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plasmid pP43NMK/ASNΔ25/B2 (23), a wapA-signal peptide was fused to the N-terminus of each loop variant, resulting in the efficient ASN secretion and little intracellular ASN activity. We thus use the extracellular fractions for further analysis. In contrast to the wild type enzyme, L5 and L6 showed enhanced extracellular ASN activity

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after expression in B. subtilis, and L6 reached the highest ASN activity (308.15 U/mL) among the loop variants (Fig.

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3a). All ASN variants were secreted by B. subtilis to comparable levels (Fig. S1a), suggesting the enhanced specific activity of L5 and L6. For further analysis, the ASN and the six variants were purified into two protein bands (40 KDa and 38 KDa) using nickel affinity chromatography on a HisTrap FF column (Fig. S1b). As indicated by MALDI-TOF mass spectrometry assays, the both bands corresponded to the ASN (data not shown). In line with extracellular ASN activity of each recombinant strain, L5 and L6 displayed enhanced specific activities while the 12

rest of the loop variants were endowed with reduced specific activities; L6 acquired the highest activity (253.43 U/mg) among the variants (Fig. 3b). Notably, the specific activity of L6 was still 7.97-fold lower than that of PcA (8), suggesting that the highly flexible N-terminal loop was one of the great causes of ASN catalytic activity.

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Flexibility analysis of N-terminal loop of B. subtilis ASN

To explore the reason for altered specific activities, RMSF values that was used to evaluate the residue flexibility

were calculated for each loop variant after MD simulations. As shown in Fig. 4a, L6 exhibited the lowest averaged RMSF value within the residues20-29 while L1 had the highest RMSF value in the same region among the loop

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variants. On the whole, RMSF values of residues20-29 negatively related to the specific activity of the ASN. These

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results suggested that reducing the flexibility of residues20-29, in the continuous non-conserved region between two

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active site residues Thr16 and Tyr30, could increase the specific activity of the ASN.

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Saturation mutagenesis at the N-terminal loop of B. subtilis ASN

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To further increase the specific activity, the saturation mutagenesis was individually performed on the two highly flexible residues (Ala26 and Gly29) of L6. After activity screening on plates, the recombinant strains expressing L6 derivatives L6-A26N (Ala26 was mutated to Asn26) and L6-G29F (Gly29 was mutated to Phe29) exhibited

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enhanced extracellular activity (Fig. 3a). The specific activities of the purified L6-A26N and L6-G29F were further

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increased as compared to L6 (Fig. 3a). Then, a double variant L6-A26N/G29F in which Ala26 and Gly29 were respectively mutated to Asn26 and Phe29 was constructed and showed further increases in extracellular ASN activity (650.33 U/mL) and specific activity (413.85 U/ mg) in contrast to the parent single-site variants of L6 (Fig. 3). Notably, all of them exhibited similar ASN bands, which indicated the yield of all variants were the same and these mutations had no effect on their expression level. (Fig S1a). Finally, the specific activity of L6-A26N/G29F 13

was 3.44-fold higher than that of wild type enzyme (Fig. 3b). MD analysis indicated that RMSF values of L6-A26N, L6-G29F, and L6-A26N/G29F were reduced in sequence (Fig. 4b). These results confirmed the inverse correlation between the flexibility of residues20-29 and ASN specific activity. Thus, reducing the flexibility of the highly flexible

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loop at N-terminus was an effective method to improve the specific activity of the enzyme.

Further catalytic characterization of the ASN loop variants

Kinetic parameters of these highly specific activity variants were determined by using L-asparagine as the substrate

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at 37°C. As presented in Table 2, contrasted to the wild-type enzyme, all variants showed a remarkable reduction in

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Km, with L6-A26N/G29F displaying the lowest value (2.8 mM), and thus the highest affinity toward L-asparagine of

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all the ASN variants. The kcat values of all variants were increased, and that of L6-G29F was almost 1.64-fold higher than wild-type ASN (Table 2). The kcat/Km values of ASN and its variants generally increased with their specific

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activities (Table 2).

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As show in Table 2, all of variants shared the same optimal temperature (65°C) with the wild-type ASN. The thermal stability of each variant was analysed at 65°C, and all thermal denaturation trails conformed to a first-order reaction. In contrast to wild type enzyme, t1⁄2 values for L6, L6-A26N, L6-G29F and L6-A26N/G29F were increased

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by 2.82, 4.02, 5.26, and 6.76-fold, respectively (Table 2). Correspondingly, the Tm values for L6, L6-A26N,

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L6-G29F and L6-A26N/G29F were 0.63, 1.26, 1.87 and 2.43°C higher than the value of 70.61°C measured for the wild-type enzyme (Table 2). To evaluate the substrate specificity, we used the L-glutamine as substrate in the same reaction conditions. As presented in Table 2, except L6, the L-glutamine activity of variants was remarkably enhanced compared with wild-type ASN, with L6-A26N, L6-G29F and L6-A26N/G29F exhibiting 276%, 314% and 371% increases in glutamine specific activity, respectively, which suggested the mutation affected the catalytic 14

efficiency of both asparagine and glutamine. For affinity purification, each ASN variant was fused with a His-tag at its N-terminus. Under the same condition, a significant deviation in ASN production and characterization was not observed between the B. subtilis

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strains expressing the modified ASN with or without the N-terminal His-tag (data not shown), indicating that the N-terminal His-tag does not affect the ASN production and characterization.

Secondary and tertiary structure analysis of the ASN loop variants

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To understand the enhanced catalytic activity, we conducted circular dichroism and fluorescence assays to analyse

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the secondary structure and tertiary structure of wild-type ASN and two highly stabilized variants (L6 and

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L6-A26N/G29F), respectively. However, Circular dichroism spectra of ASN was similar to that of the loop variants; the β-sheet and α-helical content was not altered by the mutations (Fig. 5a). In addition, the wavelength of the

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emission maximum (286 nm) was the same for wild-type and variant ASN enzymes in fluorescence spectra,

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indicating intact tertiary structure for all variants (Fig. 5b).

Interaction analysis of the ASN loop variants

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Based on the crystal structure of ErA (PDB code 1HG0), the three-dimension structure of each ASN loop variants

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was modelled. In comparison with the wild-type enzyme, three newly formed hydrogen bonds [(Gly29 (chain B)/Asp289 (chain D); Thr28 (chain B)/Asp191 (chain C); Ala26/Glu23] and a hydrophobic interaction (Ala26/Ala21) were detected in L6, and an unfavourable negative charge-charge interaction (Asp21/Gln29) (4.49 Å) was eliminated in this variant (Fig. 6). Compared with the variant L6, L6-A26N/G29F formed five new hydrogen bonds (Ser24/Gln26; Phe29/Thr28; Pro124/Thr27), among which the interactions between Ser24 and Gln26 replaced a 15

hydrogen bond (Ala26/Glu23) and a hydrophobic interaction (Ala26/Ala21) existed in L6 (Fig. 6). Moreover, three interactions were formed between the subunit B, C and D in L6-A26N/G29F, including two hydrophobic interactions between residues Phe29 (chain B) and Tyr290/Ala291 (chain D) and an electrostatic interaction between

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residues Phe29 (chain B) and Asp191 (chain C) (Fig. 6).

As indicated by sequence alignment, the N-terminal flexible loop of B. subtilis ASN contained the two highly conserved active site residues Thr16 and Tyr30, and the latter residue is critical for the correct orientation of the

former (28). The interactions between Thr16 and Tyr30 were characterized by the distances between them through

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MD trajectory. As shown in Fig. 6, the distances between the active sites Thr16 and Tyr30 in wild-type enzyme was

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higher than 3.5Å, In the case of L6 and L6-A26N/G29F, the distance was lower than 3Å. These results suggested

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that more stable hydrogen bonds between Thr16 and Tyr30 will be formed in L6 and L6-A26N/G29F in contrast to

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DISCUSSION

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wild-type enzyme.

Owing to the ability to eliminate L-asparagine, ASNs have been used as a promising antineoplastic agent and effective acrylamide-mitigating additive in food products. Generally, the effects of commercial ASNs from E. coli

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and E. chrysanthemi exhibited a dose-dependent manner (28, 29), implying that more commercial ASNs were

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required for killing more neoplastic cells. Moreover, hot water which contributes to the release of asparagine from foodstuffs (e.g. potato chips) was usually used for ASNs-mediated acrylamide degradation. Thus, to reduce the enzyme dosage and increase the application efficiency at high temperatures, it was desirable to improve the specific activity and thermal stability of ASNs. In this study, we generated a variant of B. subtilis 168 ASN, L6-A26N/G29F which showed 3.44- and 7.76-fold increases in the specific activity and t1/2 at 65°C in contrast to the wild-type 16

enzyme, respectively. The specific activity and thermal stability of L6-A26N/G29F were superior than those of the commercial ASN from the E. chrysanthemi (29). The ASN variants with enhanced activity and stability may expand the application in both medical treatment and food industry.

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It has been reported that the N-terminal highly flexible loop was critical for the activity of bacterial ASNs (28). According to sequence alignment, a continuous non-conserved region between the two active residues (Ile15 and

Thr33 in WoA) was found at the flexible N-terminal loop of these ASNs with different catalytic efficiencies (19). It was possible that this continuous non-conserved region plays a key role in function of the N-terminal flexible loop,

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and consequently affects its catalytic efficiency. However, modifications within this loop region were restricted to a

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single site, and the role of the whole sequence of the continuous non-conserved region in the enzyme activity has not

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yet been resolved (28). Based on the sequence substitution and site-saturation mutagenesis of the continuous non-conserved region (residues20-29 in B. subtilis 168 ASN), we here greatly improved the specific activity of the

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ASN. According to the activity assay and RMSF analysis of the ASN loop variants, the flexibility of the continuous

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non-conserved region in the N-terminal loop negatively related to the specific activity of ASN. This finding was consistent with the results obtained by Offman et al. (28). Thus, reducing the flexibility of the highly flexible loop at N-terminus could improve the specific activity. Notably, an excessively rigidification of the N-terminal loop may

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hinder substrate entry into the active site, inducing the decrease in the catalytic reaction (28). Therefore, further

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improvement of the ASN activity through regulating the loop flexibility need to balance the orientation of active sites and substrate entrance. Based on the mechanism for ASN catalysis, a possible explanation for negative relationship between the flexibility of the continuous non-conserved region and specific activity was that the flexibility affected the correct orientation of Thr16 that was essential for ASN reaction (16). It has been reported that the orientation of Thr16 was 17

dependent on its interaction with Tyr30 (16, 30). In contrast to wild-type enzyme, both L6 and L6-A26N/G29F showed decreased distances between the Thr16 and Tyr30, suggesting the formation of more stable interactions (hydrogen bonds) between the two active sites. Another possible explanation for the negative relationship was that

SC RI PT

the flexibility of the continuous non-conserved region directed the closure conformation of the substrate pocket. According to the findings reported by Nguyen et al. (20), the N-terminal flexible loop acting as a lid assisting

substrate binding in substrate pocket, and efficient closure of the N-terminal flexible loop are beneficial for catalytic activity. In comparison with the wild-type ASN, two additional hydrogen bonds [(Gly29 (chain B)/Asp289 (chain D);

U

Thr28 (chain B)/Asp191 (chain C)] formed between the continuous non-conserved region and the rest regions of

N

substrate pocket in L6 while L6-A26N/G29F acquired two hydrophobic interactions [Phe29 (chain B)/Tyr290 (chain

M

A

D); Phe29 (chain B)/Ala291 (chain D)] and one electrostatic interaction [Phe29 (chain B)/Asp191 (chain C)]. These findings indicated that L6 and L6-A26N/G29F displayed reduced distance between the continuous non-conserved

ED

region and the rest regions of substrate pocket and subsequently benefited the ordering and closure of the N-terminal

EP T

flexible loop.

Results from this study showed that the variant with the highest L-asparaginase activity (L6-A26N/G29F) also showed a remarkable increase in L-glutaminase activity. Notably, L6-A26N/G29F showed an increase in the

CC

interactions between N-terminal loop and substrate pocket. A similar increase was not observed in the variant L6

A

which exhibited slightly increased glutaminase activity in contrast to wild type enzyme. As demonstrated by Nguyen et al. (20), the relative low glutaminase activity was due to the weak interaction between active site Thr16 and glutamine (substrate) which hindered the substrate pocket from adopting the closed conformation. Thus, it was possible that the increase in the interactions between N-terminal loop and substrate pocket of L6-A26N/G29F promoted the closure of the substrate pocket and the consequent increase in glutaminase activity. It was generally 18

recognized that the secondary glutaminase activity accounted for the neurotoxicity of ASN (28). Thus, hurdles for the ASN variant in its practical application to medicine seem to be high owing to the enhanced glutaminase activity. To reduce the glutaminase activity, further investigation on substrate pocket should be performed.

SC RI PT

Our results also demonstrated that modification of the N-terminal loop could also benefit the thermal stability of the ASN. It was generally recognized that the introduction of these interactions (hydrogen bonds, hydrophobic

and/or electrostatic interactions, and etc.) could increase free energy barrier against the high temperature, resulting in stabilization of enzymes (31).The introduction of different interactions into the highly flexible N-terminal loop may

U

mainly account for the enhancement of L6 and L6-A26N/G29F. To be noted, the unfavourable charge-charge

N

interactions on the protein surface exert adverse effect on the thermal stability of enzymes (32, 33). As analysed by

M

A

Discovery Studio 4.0, one unfavourable charge-charge interaction (Asp21/Gln29) existed in wild-type enzyme was missed in L6 and L6-A26N/G29F, suggesting the role of elimination of the unfavourable interaction in the

ED

stabilization of the both variants.

EP T

In conclusion, a potential mechanism was proposed based on structures analysis, in which reducing the flexibility of the highly flexible loop contributes to the substrate pocket of ASN to form a closed conformation, consequently enhancing its specific activity and thermal stability. Simultaneously, ASN variants with enhanced

A

CC

specific activity and thermal stability were obtained.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31771913), Key Research and Development Program of Jiangsu Province (BE2016629).

19

Conflict of interest The authors declare that they have no conflict of interest.

SC RI PT

References

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ED

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EP T

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SC RI PT

characterization of Helicobacter pylori asparaginase, Microbiol Immunol. 55 (2011) 408-417.

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[11] S. Bansal, A. Srivastava, G. Mukherjee, R. Pandey, A.K. Verma, P. Mishra, B. Kundu, Hyperthermophilic

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mechanism of action, FASEB J. 26 (2012) 1161-1171.

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ED

B. Yao, New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28

EP T

Endo-Polygalacturonases, Plos One. 10 (2015) 16. [13] X.Y. Lu, S. Liu, Y. Feng, S.Q. Rao, X.M. Zhou, M. Wang, G.C. Du, J. Chen, Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions, Appl. Microbiol.

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A

[14] A.L. Swain, M. Jaskólski, D. Housset, J. Rao, A. Wlodawer, Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy, P Natl Acad Sci Usa. 90 (1993) 1474-1478. [15] J. Lubkowski, G.J. Palm, G.L. Gilliland, C. Derst, K.H. Röhm, A. Wlodawer, Crystal Structure and Amino Acid Sequence of Wolinella Succinogenes l‐ Asparaginase, FEBS J. 241 (1996) 201-207. [16] K. Aghaiypour, A. Wlodawer, J. Lubkowski, Structural basis for the activity and substrate specificity of Erwinia 21

chrysanthemi L-asparaginase, Biochemistry. 40 (2001) 5655-5664. [17] A.C. Papageorgiou, G.A. Posypanova, C.S. Andersson, N.N. Sokolov, J. Krasotkina, Structural and functional insights into Erwinia carotovora l‐ asparaginase, FEBS J. 275 (2008) 4306-4316.

SC RI PT

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[19] H.A. Nguyen, D.L. Durden, A. Lavie, The differential ability of asparagine and glutamine in promoting the

closed/active enzyme conformation rationalizes the Wolinella succinogenes L-asparaginase substrate specificity, Sci

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Rep. 7 (2017) 41643.

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l-asparaginase, Biochemistry. 55 (2016) 1246-1253.

N

[20] H.A. Nguyen, Y. Su, A. Lavie, Structural Insight into Substrate Selectivity of Erwinia chrysanthemi

[21] S.H. Fisher, L.V. Wray, Bacillus subtilis 168 contains two differentially regulated genes encoding

ED

L-asparaginase, J. Bacteriol. 184 (2002) 2148-2154.

EP T

[22] Y. Onishi, S. Yano, J. Thongsanit, K. Takagi, K. Yoshimune, M. Wakayama, Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties, Ann Microbiol. 61 (2010) 517-524.

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from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy, Appl. Microbiol. Biotechnol. 101 (2017) 1509-1520. [24] H. Weissig, I.N. Shindyalov, P.E. Bourne, Macromolecular structure databases: Past progress and future challenges, Acta Crystallogr D Struct Biol. 54 (1998) 1085-1094. [25] M.-K. Yun, A. Nourse, S.W. White, C.O. Rock, R.J. Heath, Crystal Structure and Allosteric Regulation of the 22

Cytoplasmic Escherichia coli l-Asparaginase I, J Mol Biol. 369 (2007) 794-811. [26] V.D. Deokar, M.D. Vetal, L. Rodrigues, Production of intracellular L-asparaginase from Erwinia carotovora and its statistical optimization using response surface methodology (RSM), Int J Chem Sci. 1 (2010) 25-36.

the principle of protein-dye binding, Anal Biochem. 72 (1976) 248-254.

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

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L-asparaginase reveals importance of dual activity for cancer cell toxicity, Blood. 117 (2011) 1614-1621.

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[29] H.A. Nguyen, Y. Su, A. Lavie, Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants

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with Diminished l-Glutaminase Activity, J Biol Chem. 291 (2016) 17664-17676.

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[30] H.P. Aung, M. Bocola, S. Schleper, K.H. Rohm, Dynamics of a mobile loop at the active site of Escherichia coli asparaginase, Biochim Biophys Acta. 1481 (2000) 349-359.

ED

[31] A.S. Bommarius, M.F. Paye, Stabilizing biocatalysts, Chem Soc Rev. 42 (2013) 6534-6565.

EP T

[32] A.V. Gribenko, M.M. Patel, J. Liu, S.A. McCallum, C. Wang, G.I. Makhatadze, Rational stabilization of enzymes by computational redesign of surface charge–charge interactions, P Natl Acad Sci Usa. 106 (2009) 2601-2606.

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[33] Y. Feng, S. Liu, Y. Jiao, Y. Wang, M. Wang, G. Du, J. Chen, Improvement of l-asparaginase thermal stability by

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regulating enzyme kinetic and thermodynamic states, Process Biochem. (2018) https://doi.org/10.1016/j.procbio.2018.05.002.

23

Figure legends Fig. 1 Sequence alignment of different ASNs. Red box represents the sequences of the N-terminus flexible loop. Green triangles indicate the active sites of ASN (Thr16 and Tyr30,). Numbering is based on B. subtilis ASN

SC RI PT

sequence (WT). WT: B. subtilis ASN (this study); EcA: E. coli ASN; HpA: H. pylori ASN; WoA: W. succinogenes ASN; PgA: P. aeruginosa ASN; ErA: E. chrysanthemi ASN; PcA: P. carotovorum ASN.

Fig. 2 The modelling structure of B. subtilis ASN. The tetramer structure was shown in surface representation.

U

Chain B, C and D were coloured grey, lemon and, green respectively. In the close-up view, the N-terminus flexible

N

loop was shown in cartoon representation and coloured red; the continuous non-conserved region (residues20-29) was

M

A

coloured cyan; the active sites Thr16 and Tyr30 were shown in stick representation and coloured red while ligand

ED

ASP was coloured yellow. Green dashed lines indicate the extended network of hydrogen bonds.

EP T

Fig. 3 Production and catalytic properties of wild-type ASN and its loop variants. (a) The extracellular ASN activity of each ASN expressed in B. subtilis; (b) The specific activity of each ASN. 110: wild-type ASN, L1, L2, L3, L4, L5, L6, L6-A26N, L6-G29F and L6-A26N/G29F, respectively. Error bars represent the average of triplicate

A

CC

experiments.

Fig. 4 RMSF profiles of wild-type ASN and its loop variants. (a) RMSF profiles of wild-type ASN and its six loop replacement variants at 300 K. (b) RMSF profiles of wild-type ASN, L6 and L6 derivatives at 300 K. After MD simulations conducted at 300 K, plots of RMSD of protein backbone were calculated for 20 ns, and all structures reached equilibrium after 12 ns of simulation. RMSF of residues536 of each subunit of ASN was calculated at 300 K 24

and then presented as an average value. The post assays were conducted using VMD.

Fig. 5 Secondary and tertiary structure analysis of wild-type and variant ASNs. (a) Secondary structure analysis

SC RI PT

of ASNs by far-UV CD spectroscopy. Spectra were obtained at 190 to 250 nm using steps of 1 nm. (b) Tertiary

structure analysis of ASNs by steady-state fluorescence spectroscopy at the same protein concentration. Emission was monitored between 250 and 330 nm using steps of 0.2 nm following excitation at 280 nm.

U

Fig. 6 Interaction analysis of wild-type and variant ASNs. Amino acids are shown in stick representation. Asn26

N

and Phe29 were coloured light cyan and green, respectively. Chain B, C and D were coloured grey, atrovirens and

M

A

dark grey, respectively. Green dashed lines indicate the extended network of hydrogen bonds. Purple and red dashed

A

CC

EP T

ED

lines indicate hydrophobic interactions and unfavourable charge-charge interaction, respectively.

25

Tables Table 1 Oligonucleotides used in this study. Sequence

F1

GGCACGATAGCTGGTGGTGGCGATTCGGCTACCAAAGCAAATTATAAAGCAGGTGTTGTC

F2

GGGCACGATAGCTGGTTCTGGTGTTGATGCTTCCTTGGGAAGCTATAAAGCAGGTGTTGTC

F3

GGGCACGATAGCTGGTTCTGGAGAATCGAGTGTCAAATCTTCTTATAAAGCAGGTGTTGT

F4

GGCACGATAGCTGGTGCTGGTGCTTCCGCTGCCAATTCCGCTACCTATAAAGCAGGTG

F5

GGCACGATAGCTGGTTCCGCTGCCACTGGTACCCAAACAACTGGATATAAAGCAGGTG

F6

GCACGATAGCTGGTAAGGCCGAATCAAATACCGCAACAACTGGATATAAAGCAGGTGTTG

A26X

AGGCACGATAGCTGGTAAGGCCGAATCAAATACCNNKACAACTGGATATAAAGCA

G29X

GCACGATAGCTGGTAAGGCCGAATCAAATACCGCAACAACTNNKTATAAAGCAGGTGTT

M

A

N

U

SC RI PT

Primers

29F

ED

A26N/G

R1

ACCAGCTATCGTGCCTCCTGTCGCTAAAATTCT

EP T

GCACGATAGCTGGTAAGGCCGAATCAAATACCAACACAACTTTTTATAAAGCAGGTGTT

Notes: the underlines indicate the mutation sites; X represented any of amino acid; N represented any of the

A

CC

following A, T, G, or C; K represented G or T; M represented A or C.

26

Table 2 Enzymatic properties of the wild-type ASN and its loop variants. kcat

Km

kcat/Km

Topt

t1/2 (65 °C)

GLN specific Tm (°C)

(sec-1)

(mM) (sec-1·mM-1) (°C)

(min)

WT

39.59

5.97

6.63

65

58.54

70.61

L6

46.26

4.49

10.29

65

223.52

71.24

L6-A26N

36.90

2.81

13.13

65

294.15

71.87

L6-G29F

65.00

3.95

16.44

65

366.44

72.48

L6-A26N/G29F 53.72

2.80

19.18

65

454.78

73.04

activity (U/mg) 2.05

SC RI PT

Enzyme

2.10 5.65

N

U

6.44 7.61

A

CC

EP T

ED

M

A

Notes: Each value is the mean of triplicate experiments, and the variation about the mean is below 5 %.

27

SC RI PT

Figures

N-terminus flexible loop

A

CC

EP T

ED

M

A

N

U

(Fig. 1)

28

N-terminus flexible loop

Tyr30

SC RI PT

Thr16

ligand ASP

A

CC

EP T

ED

M

A

N

U

(Fig. 2)

29

ED

EP T

CC

A

SC RI PT

(Fig. 3)

U

N

A

M

a b

30

ED

EP T

CC

A (Fig. 4)

SC RI PT

U

N

A

M

a b

31

ED

EP T

CC

A (Fig. 5)

SC RI PT

U

N

A

M

a b

32

WT E29 Q22 D21

D191” S27

Y30

P124

3.6Å T16

L6

U

L6-A26N/G29F E23

N A

N26

D191” T27 T28

P124

D191”

EP T

T28

2.9Å

T16

A

CC

(Fig. 6)

ED

G29

T16

A291’

F29

M

D289’

A26

Y30

Y290’

S24

A21

P124

SC RI PT

T28

33

Y30

2.7Å