KR102421107B1 - Acyl-CoA thioesterase derived from Bacillus cereus and variants thereof - Google Patents

Abstract

As a variant of acyl-CoA thioesterase derived from Bacillus cereus, acyl-CoA thioesterase variant in which serine, aspartic acid, or a combination thereof located at the binding site to the adenine moiety of CoA is substituted with alanine is initiated.

Description

ACT derived from Bacillus cereus and variants thereof {Acyl-CoA thioesterase derived from Bacillus cereus and variants thereof}

The present invention relates to ACT derived from Bacillus cereus and variants thereof.

Acyl-CoA thioesterase (Acyl-CoA thioesterase, hereinafter may be referred to as ACT) is an enzyme that produces fatty acids by cleaving between a sulfur atom and a carbonyl group in a thioester bond (see FIG. 1). ACT is an intracellular It plays an important role in lipid metabolism and cellular function by regulating the concentration of CoA, fatty acids, and acyl-CoA, the sequence of which is highly conserved across bacteria, fungi, plants, and mammals. Furthermore, acyl-CoA plays an important role in the regulation of ion channels, various signal transduction pathways, budding and fusion of the cell inner membrane, and allosteric regulation of various enzymes such as acetyl-CoA carboxylase and hexokinase IV. Therefore, physiologically, the role of ACT is very important.

ACT is largely divided into two groups according to amino acid sequence and protein folding. Type I ACT belongs to the α/β-hydrolase fold enzyme superfamily and is only found in animals. Type I ACTs have an N-terminal β-sandwich domain and a C-terminal α/β-hydrolase domain. A catalytic triad composed of Ser294, Asp388, and His422 is located in a loop connecting the α-helix and the β-strand of the C-terminal α/β-hydrolase domain.

In contrast, type II ACT belongs to the hotdog-fold enzyme superfamily. Type II ACT exists in a variety of organisms, including bacteria, fungi, and plants, and various catalytic residues and mechanisms have been reported because the substrate-binding pocket and catalytic residues are not conserved. Catalysis of type II ACT generally requires dimerization of two hot dog domains, with catalytically active sites located at the dimer interface. Interestingly, the composition of the double hotdog domain of ACT is highly variable in prokaryotes and eukaryotes. In prokaryotes, predominantly a single polypeptide forms a hot dog domain and the same two polypeptides constitute an active dual hot dog domain. However, in eukaryotes, one polypeptide forms two distinct hot dog domains. This difference in dimer composition determines the number of active sites in the dual hot dog domain. ACT in prokaryotes forms homohexamers, whereas eukaryotes form trimers in heterodimers. Prokaryotes have two active sites per one double hot dog domain, whereas eukaryotes have one active site per one double hot dog domain.

Bacillus cereus is a Gram-positive, facultative anaerobic, spore-producing, motile and rod-shaped bacterium. Bacillus cereus has two ACT (BC_5426 and BC_2038) genes, among which the structure of BC_2038 is known (refer to PDB code 1Y7U). It is not known about variants capable of increasing it.

Republic of Korea Publication No. 10-2019-0141683 (2019.12.24)

According to one embodiment, as a variant of acyl-CoA thioesterase derived from Bacillus cereus, a variant having increased catalytic activity is provided.

One aspect is a variant of acyl-CoA thioesterase derived from Bacillus cereus, in which serine, aspartic acid, or a combination thereof located at the binding site to the adenine moiety of CoA is substituted with alanine. Terase variants are provided.

The Bacillus cereus strain has two ACT candidate genes, and the present inventors confirmed that they express type II acyl-CoA thioesterase from BC_5426. The Bacillus cereus may be strain ATCC 14579, and anyone can receive it from atcc.org.

It was confirmed that the monomer of the Bacillus cereus-derived acyl-CoA thioesterase (BcACT1) has a hot dog folding structure, and forms a hexameric structure in which three dimers are bonded. According to one embodiment, the adenine ring of CoA is located deep in the binding pocket of BcACT1 and can be stabilized by Ser57, Asp62, and Arg152. The present inventors confirmed that the activity was rather increased when Ser57 and Asp62, which were expected to stabilize the adenine ring of CoA, were mutated to alanine during the experiment by producing various mutants to identify residues related to CoA binding.

According to one embodiment, the serine, aspartic acid, or a combination thereof may interact with the adenine moiety of CoA.

According to one embodiment, the acyl-CoA thioesterase may consist of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is an acyl-CoA thioesterase (BcACT1) sequence of strain ATCC 14579, which is a reference strain of Bacillus cereus.

According to one embodiment, the serine may be serine at position 57 in SEQ ID NO: 1, and the aspartic acid may be aspartic acid at position 62 in SEQ ID NO: 1.

The mutant can be prepared by site-directed mutagenesis or mutagenesis generated by PCR according to methods known in the art.

Another aspect provides an expression vector comprising a polynucleotide encoding the acyl-CoA thioesterase variant.

The expression vector may include a promoter for regulating the expression of the encoded protein. The promoter may be, for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter. The expression vector may include RBS for initiation of translation and transcription/translation termination sequences. In addition, when E. coli is used as a host cell, the promoter and operator sites of the E. coli tryptophan biosynthetic pathway and the leftward promoter (pLλ promoter) of phage λ may be included as regulatory regions.

Vectors that can be used in the present invention include plasmids often used in the art (eg, pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1 and M13). etc.) or a virus (eg, SV40, etc.), but is not limited thereto.

Another aspect provides a transformed strain comprising the expression vector.

The strain may be a transformant transformed with an acyl-CoA thioesterase mutant expression vector.

As the host cell for producing the transformant, a host cell known in the art may be used, for example, E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, Bacillus genus strains such as Bacillus cereus, and Salmonella typhimurium, Serratia marcesens and various Pseudomonas species The same enterobacteriaceae and strains may be used, but the present invention is not limited thereto.

As a method of transporting a vector or the like into a host cell in the transformation process, the CaCl2 method (Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)), Hanhan method (Cohen, S.N. et al., Proc. Natl. Acac.Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580 (1983)) or An electroporation method may be used, but is not particularly limited.

The strain can have a variety of uses due to the increased activity of the acyl-CoA thioesterase variant. For example, the transformed strain may be used for biofuel, surfactant, or biodegradable bioplastic precursor production by increasing free fatty acid production by itself or by introducing a mutation, but is not limited thereto.

The culture conditions of the strain of the transformed strain may vary depending on the host cell, but in general, the temperature is 25 to 40 °C, and the pH is preferably 6 to 10.

The variant of acyl-CoA thioesterase derived from Bacillus cereus according to one embodiment may have higher activity than WT.

The mutant expression strain of acyl-CoA thioesterase according to one embodiment may increase the production of free fatty acids.

1 shows the enzymatic reaction of Acyl-CoA thioesterase. In Acyl-CoA, the red line indicates the region where the enzymatic reaction occurs.
Figure 2 shows the amino acid sequence alignment results of ACT. The secondary structure is for the BcACT1 structure. Catalyst residues are indicated by red triangles. Residues involved in CoA binding are indicated by green triangles. Residues with increased activity upon mutation to alanine are indicated by green circles. The β1-α1 linkage loop, which undergoes conformational changes upon substrate binding, and the additional α2 helix, which determines the mechanism of nucleotide regulation, are shown in cyan and yellow, respectively. BcACT1 and BcACT2 represent ACT1 and ACT2 of Bacillus cereus ATCC 14579, respectively. SaACT and NmACT are from Staphylococcus aureus subsp. ACT of Aureus Mu50 and Neisseria meningitides is shown. MmACT_N and MmACT_C represent the N-terminal domain and C-terminal domain of ACT derived from Mus musculus, respectively. HsACT_N and HsACT_C represent the N-terminal domain and C-terminal domain of ACT derived from Homo sapiens, respectively.
3 is a monomer (monomer) structure of BcACT1. The secondary structural elements are distinguished by different colors. The picture on the right is a horizontal rotation of the picture on the left by 90 degrees.
4 shows the oligomeric structure of BcACT1.
4A is a hexameric structure of BcACT1. Six molecules were distinguished by different colors. CoA bound to BcACT1 is indicated by the magenta stick model.
4B is a size exclusion chromatography result of BcACT1. a to d show standard samples of thyroglobulin (669 kDa), aldolase (158 kDa), conalbumin (75 kDa), and carbonic anhydrase (29 kDa), respectively.
4C is a dimer structure of BcACT1. The two molecules are distinguished by different colors. Bound CoA is represented by the magenta stick model.
5 shows the active site of BcACT1.
Figure 5A shows the overlapping structure of the catalytic site of BcACT1 and SaACT. The catalytic residue of BcACT1 is indicated by a cyan bar, and the catalytic residue of SaACT is indicated by an orange bar. The β1-α1-connected loops of BcACT1 and SaACT are shown in blue-green and orange, respectively. CoA bound to BcACT1 and butyryl-CoA bound to SaACT were indicated by stick models in magenta and light blue.
Figure 5B is the entrance to the butyryl group binding pocket of SaACT. The SaACT structure is shown as a surface model, and the β1-α1 linked loop region is shown in orange.
Figure 5C is the entrance to the butyryl group binding pocket of BcACT1. The BcACT1 structure is shown as a surface model, and the β1-α1 linked loop region is shown in cyan. The butyryl-CoA molecule is derived from the SaACT structure and is shown in light blue.
5D shows the CoA binding method of BcACT1. Each monomer is distinguished by its gray and light pink colors. CoA molecules are represented by the magenta stick model. Residues of gray monomers are shown in cyan and residues of light pink monomers are shown in green. Hydrogen bonds and salt bridges are indicated by red dotted lines.
5E is a result of a site-directed mutagenesis experiment of BcACT1. The experiment was performed three times.
5F shows the adenine binding pockets of BcACT1 ^WT and three BcACT1 variants. The structure of BcACT1 was represented by an electrostatic potential surface model, and CoA was represented by a stick model of magenta color. The structures of the three variants were prepared by mutating the residues using PyMOL. The positions of Ser57 and Asp62 are indicated by yellow and red circles, respectively.
6 is a result of confirming whether or not the nucleotide-independent regulation of BcACT1.
6A is a result confirming the activity of BcACT1 by adding various concentrations of GDP or ADP. The experiment was performed three times.
6B and 6C show blockade of nucleotide binding in BcACT1. The structure of BcACT1 is shown as a cartoon model in FIG. 6B and a surface model in FIG. 6C. The NmACT structure overlapped with BcACT1, and the GDP molecule bound to NmACT is indicated by a light blue bar model. Residues located at the nucleotide binding site of NmACT are indicated by a magenta stick model. The α2-helix is indicated in cyan.
6D is a result of overlapping nucleotide-independent ACT structures. The structures of BcACT1, BcACT2, and SaACT were overlapped with GDP-coupled NmACT and ADP-coupled HsACT. Combined GDP and ADP molecules are represented by light blue and orange bar models, respectively. The α2-helices of BcACT1, BcACT2, and SaACT1 are indicated in cyan, green, and yellow, respectively.
6E is a structural overlap result of nucleotide-dependent ACT. The structures of NmACT, MnACT, and HsACT were superimposed, and their α2-helices were indicated in light blue, magenta, and orange, respectively.
6F is a surface model of the NmACT structure. The α2-helix is shown in light blue.

Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Experimental method

1. BcACT1 Preparation

The BcACT1 gene was amplified by PCR using the chromosomal DNA of Bacillus cereus ATCC 14579 as a template. The PCR product was cloned into the pET30a expression vector. The pET30a:BcACT1 vector was introduced into the E. coli strain BL21(DC3)-T1 ^R for transformation, and cultured in 0.5L terrific broth medium containing kanamycin at 37°C. When the OD ₆₀₀ reached 1.0, 0.5 mM IPTG was added to induce BcACT1 protein expression. After 20 hours at 18°C, cells were harvested by centrifugation at 4000xg at 20°C for 15 minutes. The cell pellet was resuspended in Buffer A (40 mM Tris-HCl, pH 8.0) and disrupted by sonication. Cell debris was removed by centrifugation at 13000xg for 30 minutes, and the lysate was applied to buffer A and an equilibrated Ni-NTA agarose column (Quiagen). After washing with buffer A containing 25 mM imidazole, the binding protein was eluted with buffer B (40 mM Tris-HCl, pH 8.0 and 300 mM imidazole). Finally, trace contaminants were removed by size-exclusion chromatography using Buffer A and an equilibrated HiPrep 26/60 Sephacryl S-300 HR column (320 mL, GE Healthcare Life science). All purification experiments were performed at 4°C.

2. Crystallization of BcACT1

Crystallization of purified BcACT1 was performed with sparse-matrix screens including Index, PEG ion I and II (Hampton Research), and Wizard Classic I and II (Rigaku Reagents). A sitting-drop vapor-diffusion method was performed at 20 °C. Each experiment consisted of mixing 1.0 μl protein solution (51.8 mg/ml at 40 mM Tris-HCl, pH 8.0) with 1.0 μl reservoir solution and equilibrating against 500 μl reservoir solution. BcACT1 crystals were observed under several crystallization screening conditions. Following several steps using the hanging-drop vapor-diffusion method for crystal improvement, the highest quality crystals were obtained in 0.49 M sodium phosphate monobasic monohydrate and 0.91 M potassium phosphate dibasic. could

3. BcACT1 structure determination (structure determination)

Crystals of BcACT1 were transferred to a cryo-protectant. Crystals were harvested using loops larger than crystals and flash frozen in nitrogen at -173°C.

Data were collected at a resolution of 2.89 Å on the 7A beamline of the Pohang Accelerator Laboratory (PAL, Pohang, Korea) using a Quantum 270 CCD detector (ADSC, USA). All data were indexed, consolidated and expanded using the HKL-2000 software package.

The crystals of BcACT1 belonged to spatial group P 31 with unit cell parameters a = 78.685 Å , b = 78.685 Å , c = 215.63 Å , α = 90°, β = 90° and γ = 120°. Assuming 6 BcACT1 molecules per asymmetric unit, the crystal volume per protein mass unit was 3.37 Å ³ Da ⁻¹ , indicating a solvent content of 63.50%. The structure of BcACT1 was determined by molecular replacement of the CCP4 version of MOLREP using the structure of another Acyl-CoA thioesterase from Bacillus cereus ATCC 14579 (PDB code 1Y7U) as a search model. I used the WinCoot program to manually build the model and made improvements in CCP4 refmac5. Data statistics are presented in Table 1 below. The purified model was deposited with the Protein Data Bank as a PDB code of 7CZ3.

4. Size exclusion chromatography analysis of BcACT1

To confirm the oligomeric state of BcACT1, analytical size exclusion chromatography was performed using a Super growth 200 10/300 GL column (GE Healthcare Life Sciences) equilibrated with 40 mM Tris-HCl, pH 8.0 and 150 mM NaCl. 1 ml of protein sample with a concentration of 1 mg/ml was used for analysis. To calculate the molecular weight of the eluted BcACT1 sample, a calibration curve was used as a standard sample for thyroglobulin (669 kDa), aldolase (158 kDa), and conalbumin (75 kDa). and carbonic anhydrase, 29 kDa) (GE Healthcare Life Sciences).

5. BcACT1 Activity Assay

Site-specific mutations were generated using the Quick Change Kit (Stratagene) and sequencing was performed to confirm the correct integration of the mutations. The mutant protein was purified in the same manner as wild-type BcACT1. Acyl-CoA thioesterase catalyzes acyl-CoA as a substrate to form free CoA (free CoA) with fatty acids. The activity of BcACT1 was measured using the reaction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent). DTNB reacts with free CoA to form 5-thio-2-nitrobenzoate, which absorbs light at 412 nm. Activity assays were performed at room temperature with a total volume of 500 μL of the reaction mixture. The reaction mixture contained 0.1 M sodium phosphate, pH 7.4, 1 mM acetyl-CoA and 1 mM DTNB. Reactions were started by adding wild-type or mutant BcACT1 protein to a final concentration of 2 μM. Analysis with GDP or ADP added was performed in the same manner as described above, except that various concentrations of GDP or ADP were included in the reaction mixture.

Example 1: Overall structure of BcACT1

To understand the molecular mechanism of BcACT1, the crystal structure was determined at a resolution of 2.89 Å. The overall structure of BcACT1 is Bacillus cereus ACT2 (BcACT2, PDB code 1Y7U), Staphylococcus aureus subsp. Aureus Mu50 ACT (SaACT, PDB code 5EGL), Neisseria meningitidis ACT (NmACT, PDB code 5V3A), Mus musculus ACT (MmACT, PDB code 2Q2B and 2V1O) and Homo sapiens ACT (HsACT, PDB code 4MOB) of various organisms The structure was similar to that of type II acyl-CoA thioesterases. (Fig. 2)

The monomeric structure of BcACT1 exhibits a hot dog-like structure and consists of a central α-helix (α1) and a 5-stranded antiparallel β-sheet (β1-β5) surrounding the α-helix. (See Fig. 3) In terms of shape, α-helix can be compared to a hot dog sausage, and β-sheet can be compared to a bun. BcACT1 also has an additional α-helix (α2) at the C-terminus. The α2 helix is located opposite the β-sheet and exhibits a dish-like structure. The structural analysis confirmed that BcACT1 belongs to type II acyl-CoA thioesterase.

The asymmetric unit of crystal contains six BcACT1 molecules and forms a hexameric structure consisting of a trimer of three dimers (FIG. 4A). Analytical size exclusion chromatography was performed to confirm the oligomeric structure of BcACT1 in solution. The eluted protein showed a molecular weight of about 108.07 kDa, indicating that BcACT1 was present as a hexamer in solution (Fig. 4B). In the case of the dimer, the β2 and β2-β3 connecting loops and central α-helix (α1) of each monomer were confirmed to be the main structures of the double hotdog domain (Fig. 4C). Two salt bridges and six hydrogen bonds were formed at the dimer interface between the main chain of each β2 and the loop connecting β2 and β3. Three salt bridges and three hydrogen bonds were formed at the interface between the two double hot dog domains in the trimer of the three dimers.

Example 2: Active site of BcACT1

Although no additional chemical compound was added during purification and crystallization, a strong CoA electronic map was observed, allowing us to guess the active site of the enzyme. MmACT belongs to the TE6 family of 25 ACT families and has been reported to use Asn and Asp residues for enzymatic catalysis. The structure of BcACT1 bound to CoA includes Asn23 and Asp38 residues near the thiol group of the bound CoA molecule. (FIG. 5A) Therefore, it is possible that the enzymatic catalysis of BcACT1 can occur in the same way as in MmACT, so it is possible that BcACT1 also belongs to the TE6 family. In order to confirm the relationship between Asn23 and Asp38 residues and enzyme catalysis, the above two residues were substituted with alanine. When the enzymatic activity of these variants was compared with the wild type, both BcACT1N23A and BcACT1D38A mutants showed complete loss of enzymatic activity. (FIG. 5E) These two residues are also conserved in other structurally similar acyl-CoA thioesterases such as BcACT2, SaACT, NmACT and HsACT. Therefore, it was confirmed that BcACT1 belongs to the TE6 family. (See Fig. 2)

Example 3: BcACT1 substrate binding site

To identify the acyl group binding pocket of BcACT1, the BcACT1 structure complexed with a CoA molecule was superimposed with the SaACT structure complexed with butyryl-CoA. The thiol group of CoA faces inward of the BcACT1 structure, whereas the butyryl group faces outward in SaACT (Fig. 5A). This difference is thought to be due to the absence of an acyl group in the ligand bound to BcACT1, and in BcACT1, the acyl group on the substrate If there is a group, it is expected that it can be positioned in a manner similar to that in SaACT.

Interestingly, a large structural difference was observed in the linkage between β1 and α1 in which the butyryl group is located (Fig. 5A). The conformation of this region of SaACT (the linkage between β1 and α1 in which the butyryl group is located) is butyryl-CoA Allows the binding pocket to open wide outside the protein. (FIG. 5B) Accordingly, it is speculated that SaACT can accept acyl-CoA with a longer chain in its pocket and use it as a substrate. In contrast, the corresponding region of BcACT1 is located 4.2 Å (Fig. 5A) apart from that of SaACT, resulting in a closed acyl group binding pocket (Fig. 5C). However, residues constituting the BcACT1 and SaACT regions are highly conserved with each other (Fig. 2), and the b-factor of the BcACT1 region is relatively higher than the average b-factor. Based on these observations, it is predicted that BcACT1 and SaACT may have open-closed conformational changes in the acyl-CoA binding pocket upon substrate binding.

The CoA binding site is located at the interface of the BcACT1 dimer. According to FIG. 5D, in the dimer, one monomer mainly contributes to the formation of sites and the other monomer provides structural assistance. The pantothenic acid moiety of CoA is mostly stabilized by hydrogen bonding with the main chain of the enzyme, whereas the diphosphate moiety forms a hydrogen bond with the Arg152 residue. The deoxyribose phosphate moiety forms a strong hydrogen bond network with Lys84 of the main chain as well as Ser85 and Ser86 of the side chain. In addition, the adenine ring is located deep in the pocket and is stabilized by Ser57, Asp62 and Arg152 of the side chains. Most of the residues involved in CoA binding are provided by one monomer with the exception of the Asp62 residue.

To identify residues involved in CoA binding, the above-mentioned residues were replaced with alanine, and the activity of the mutant was compared with that of the wild type. According to Figure 5E, BcACT1 S85A, BcACT1 S86A and BcACT1 R152A mutants showed a dramatic decrease or almost complete loss of activity, confirming that these residues are important for stabilization of the diphosphate and phosphate moieties. In contrast, the Lys84 to alanine mutation did not affect the enzyme activity, thus concluding that the side chain of Lys84 is not involved in the stabilization of the phosphate moiety. Surprisingly, the BcACT1 S57A and BcACT1 D62A mutants exhibited dramatically increased activity (approximately 5-fold and 9-fold, respectively) compared to BcACT1 WT. Replacing the Ser57 and Asp62 residues with alanine may structurally optimize the binding site for the adenine ring ( FIG. 5F ).

How the combination of the two mutations BcACT1 S57A and BcACT1 D62A affects enzyme activity was investigated. According to Fig. 5E, the BcACT1 S57A/D62A mutant showed a 10-fold increase in activity compared to BcACT1WT, suggesting that double mutation of Ser57 and Asp62 residues resulted in a synergistic effect in the optimization of the adenine ring binding site. (See Fig. 5F)

Example 4: Structural basis for nucleotide-independent regulation of BcACT1

Some ACTs are known to be regulated by GDP or ADP nucleotides. For example, NmACT is regulated by GDP whereas HsACT is regulated by ADP/ATP. To determine whether BcACT1 is regulated by GDP or ADP, we performed an activity assay by adding GDP and ADP molecules. However, no change in the enzymatic activity of BcACT1 was observed regardless of the added nucleotide concentration ( FIG. 6A ).

These results indicate that, unlike NmACT and HsACT, BcACT1 is not regulated by GDP or ADP. To investigate the structural basis of nucleotide-independent regulation of BcACT1, the structure of BcACT1 was compared with that of other ACT homologues. The overall structure and active site were generally conserved, but substantial structural differences were observed in the nucleotide binding site. In NmACT and HsACT, the nucleotide binding site is located at the dimer interface and mainly consists of an additional α2 helix of two monomers. GDP molecules bound to NmACT interact directly with residues of Arg5, Asn81, Arg93, Ser109, Tyr111, Arg138, Lys141, Asp155 and Ser157. ADP molecules bound to HsACT are stabilized by residues of Ser238, Asn252, Arg264, Arg312 and Arg313. However, when the structure of BcACT1 was overlapped with the structure of the nucleotide complex of NmACT and HsACT, the optimized nucleotide binding site in BcACT1 could not be confirmed (B, C, F of FIG. 6).

Instead, we observed that BcACT1 had hydrophobic and bulky residues such as Phe78, Phe90, Trp136, Met158 and Leu162 located at the positions of residues involved in nucleotide binding in NmACT and HsACT (Figure 6B). These hydrophobic residues It is speculated that they interfere with the binding of BcACT1 to GDP or ADP molecules. A large structural difference was also observed in the additional α2-helix. NmACT and HsACT, known to be nucleotide-regulated, have α2-helices of length and shape optimized for nucleotide accommodation. (FIG. 6E) NmACT has a relatively short α2-helix with 24 amino acids to provide space for nucleotide binding. HsACT has a long α2 helix of 29 amino acids, but the helix is bent and has the same effect as a short α2 helix. In contrast, BcACT1 has a straight long α2-helix with 33 amino acids, which appears to completely block nucleotide binding (Figure 6D). It defines the structure of the site and is important in determining the mode of regulation of ACT by nucleotides. These structural observations make it possible to speculate whether nucleotide-dependent regulation of other ACTs has not been reported. For example, MmACT has a short α2 helix of 24 amino acids (Fig. 6E) and BcACT2 and SaACT have a straight long α2 helix of 30 and 34 amino acids, respectively (Fig. 6D). Thus, MmACT may be nucleotide-regulated, whereas BcACT2 and SaACT may not.

In this study, the crystal structure of BcACT1, enzyme catalysis, and residues involved in substrate binding were identified. We also found that BcACT1's activity was not regulated by nucleotides, suggesting that the length and shape of the additional α2-helix are key constructs that determine whether regulation is nucleotide-dependent or independent.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Acyl-CoA thioesterase derived from Bacillus cereus and variants it <130> PN200311 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> BcACT1 <400> 1 Met Glu Lys Lys Phe Met Arg Glu Ser Lys Ala Ile Lys Thr Thr Arg 1 5 10 15 Val Phe Pro Asn Asp Leu Asn Asn His Gln Thr Leu Phe Gly Gly Lys 20 25 30 Leu Leu Ala Glu Ile Asp Ser Ile Ala Ser Ile Ala Ala Ala Arg His 35 40 45 Ser Arg Lys His Cys Val Thr Ala Ser Ile Asp Ser Val Asp Phe Leu 50 55 60 Thr Pro Ile His Gln Ala Asp Ser Val Cys Tyr Glu Ala Phe Val Cys 65 70 75 80 Tyr Thr Gly Lys Ser Ser Met Glu Val Phe Val Lys Val Ile Ala Glu 85 90 95 Asn Leu Leu Val Gly Glu Arg Arg Ile Ala Ala Thr Cys Phe Ile Thr 100 105 110 Phe Val Ala Leu Lys Asp Gly Lys Pro Ser Ser Val Pro Gln Val Leu 115 120 125 Pro Glu Thr Glu Glu Glu His Trp Leu His Thr Thr Gly Ser Glu Arg 130 135 140 Ala Glu Asn Arg Arg Lys Gly Arg Leu Lys Ser Lys Glu Met Ala Glu 145 150 155 160 Val Leu Thr Leu Ser Lys Pro Trp Asn Met 165 170 <210> 2 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> BcACT1 variant <220> <221> VARIANT <222> (57) <223> Xaa (57) = Ser or Ala <220> <221> VARIANT <222> (62) <223> Xaa (67) = Asp or Ala <400> 2 Met Glu Lys Lys Phe Met Arg Glu Ser Lys Ala Ile Lys Thr Thr Arg 1 5 10 15 Val Phe Pro Asn Asp Leu Asn Asn His Gln Thr Leu Phe Gly Gly Lys 20 25 30 Leu Leu Ala Glu Ile Asp Ser Ile Ala Ser Ile Ala Ala Ala Arg His 35 40 45 Ser Arg Lys His Cys Val Thr Ala Xaa Ile Asp Ser Val Xaa Phe Leu 50 55 60 Thr Pro Ile His Gln Ala Asp Ser Val Cys Tyr Glu Ala Phe Val Cys 65 70 75 80 Tyr Thr Gly Lys Ser Ser Met Glu Val Phe Val Lys Val Ile Ala Glu 85 90 95 Asn Leu Leu Val Gly Glu Arg Arg Ile Ala Ala Thr Cys Phe Ile Thr 100 105 110 Phe Val Ala Leu Lys Asp Gly Lys Pro Ser Ser Val Pro Gln Val Leu 115 120 125 Pro Glu Thr Glu Glu Glu His Trp Leu His Thr Thr Gly Ser Glu Arg 130 135 140 Ala Glu Asn Arg Arg Lys Gly Arg Leu Lys Ser Lys Glu Met Ala Glu 145 150 155 160 Val Leu Thr Leu Ser Lys Pro Trp Asn Met 165 170

Claims (6)

As a variant of acyl-CoA thioesterase derived from Bacillus cereus,
An acyl-CoA thioesterase variant in which the serine at position 57, the aspartic acid at position 62, or a combination thereof is substituted with alanine of an acyl-CoA thioesterase consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1,
wherein the serine, aspartic acid, or a combination thereof interacts with the adenine moiety of CoA,
Acyl-CoA thioesterase variants. delete delete An expression vector comprising a polynucleotide encoding the acyl-CoA thioesterase variant of claim 1. A transformant strain comprising the expression vector of claim 5 .

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