Terminal deoxynucleotidyl transferase mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology, and specifically relates to a terminal deoxynucleotidyl transferase mutant and its application. The mutant is obtained by subjecting any one of the amino acid sequences shown in SEQ ID NOs. 1 to 7 to R189L / K191G mutations. The present invention starts with a truncated terminal deoxynucleotidyl transferase TdT from Zonotrichia albicollis, fixes 52 key residues located in the substrate binding pocket, and obtains seven terminal deoxynucleotidyl transferase mutant sequences, five of which have an unfolding temperature T m The temperature is more than 10°C higher than that of the wild type WT / LG. After the obtained mutant is heat-treated, its activity is characterized. Compared with the wild type, the mutant obtained by the present invention can significantly extend the primer and has a higher product yield, which can play an important role in DNA enzymatic synthesis.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant and application thereof. Background Art

[0002] Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of randomly adding deoxynucleotides to initiators under template-independent conditions. This template-independent nature allows TdT to be used in DNA synthesis. Using 3′-ONH2-modified nucleotides enables controlled DNA addition. However, when the growing single-stranded DNA molecule reaches a certain length, secondary structures form. TdT has poor activity with substrates that form secondary structures at the 3′-OH terminus, resulting in reduced extension efficiency. High temperatures can disrupt the hairpin structure of the primer strand, restoring it to a single strand that is easily extendable. However, TdT is unstable and can lose its enzyme activity and catalytic function at high temperatures. Therefore, modifying TdT to improve its thermal stability is of great significance in enzymatic DNA synthesis.

[0003] Traditional protein engineering relies primarily on rational design, semi-rational design, and directed evolution. Directed evolution uses methods like error-prone PCR and DNA shuffling to randomly generate large mutant libraries and screen for target mutants. Directed evolution does not require knowledge of the protein's catalytic mechanism or structure. Through multiple rounds of screening, beneficial mutations are accumulated to obtain mutants with specific performance improvements. Semi-rational design uses methods like iterative saturation mutagenesis (ISM) and combinatorial active-site saturation test (CAST) to select a few sites for modification, requiring only a smaller mutant library to be screened. Rational design relies on the relationship between protein structure and function. Through methods like molecular docking and molecular dynamics simulations, changes in protein structure and binding energy are assessed. Mutants that meet the requirements are selected and validated experimentally, significantly reducing the number of libraries required. However, traditional protein engineering suffers from low efficiency and relies on modifications to existing proteins in nature, making it unsuitable for industrial production.

[0004] In recent years, deep learning has demonstrated tremendous potential in protein design and engineering. For example, AlphaFold2 can predict protein structure, significantly accelerating protein research. Furthermore, given a protein backbone, deep learning algorithms can rapidly generate candidate amino acid sequences. ProteinMPNN, a deep learning-based protein design algorithm, can quickly and efficiently generate protein sequences with desired functions and structures, and has demonstrated success in increasing protein expression and stability. Summary of the Invention

[0005] In order to address the drawback of poor heat resistance of terminal deoxynucleotidyl transferase in the prior art, the present invention provides a terminal deoxynucleotidyl transferase mutant with high heat stability and its application. The specific technical solution is as follows:

[0006] In a first aspect, the present invention provides a terminal deoxynucleotidyl transferase mutant, wherein the terminal deoxynucleotidyl transferase mutant is one of the following mutants:

[0007] (1) The amino acid sequence shown in SEQ ID NO. 1 was obtained by subjecting it to R189L / K191G mutation;

[0008] (2) The amino acid sequence shown in SEQ ID NO. 2 was obtained by subjecting it to R189L / K191G mutation;

[0009] (3) The amino acid sequence shown in SEQ ID NO. 3 was obtained by subjecting it to R189L / K191G mutation;

[0010] (4) The amino acid sequence shown in SEQ ID NO. 4 was obtained by subjecting it to R189L / K191G mutation;

[0011] (5) The amino acid sequence shown in SEQ ID NO.5 was subjected to R189L / K191G mutation.

[0012] The present invention uses AlphaFold2 to predict the three-dimensional structure of the N-terminally truncated ZaTdT (sZaTdT, WT) and uses it as input for ProteinMPNN. Since ProteinMPNN is a structure-based de novo design method, it does not consider the functional information of the protein when generating the sequence. Therefore, in order to retain the catalytic activity of TdT, when using ProteinMPNN to design new sequences, the 52 key residues located in the substrate binding pocket are fixed. Ultimately, 7 initial terminal deoxynucleotidyl transferase mutant sequences were obtained, and AlphaFold2 was used to predict the structures of these 7 sequences. The results showed that they all reproduced the substrate binding pocket with high confidence (plDDT). The R189L / K191G mutations were combined with seven sequences to obtain terminal deoxynucleotidyl transferase mutant sequences. Terminal deoxynucleotidyl transferase mutants with improved thermal stability were obtained through expression and purification. It was verified that five of the terminal deoxynucleotidyl transferase mutants had significantly improved thermal stability. After heat treatment at 48°C, 51°C, 56°C, 58°C, and 61°C, the yield of DNA synthesis catalyzed by these five terminal deoxynucleotidyl transferase mutants was higher than that of the N-terminally truncated terminal deoxynucleotidyl transferase with the R189L / K191G mutation.

[0013] In a second aspect, the present invention provides a gene encoding the terminal deoxynucleotidyl transferase mutant.

[0014] In a third aspect, the present invention provides a recombinant vector comprising the encoding gene as claimed in claim 2.

[0015] In a fourth aspect, the present invention provides a genetically engineered bacterium, comprising a host cell, wherein the host cell contains the encoding gene according to claim 2 or the recombinant vector according to claim 3.

[0016] Furthermore, the host cell is Escherichia coli.

[0017] Furthermore, the host cell is E. coli BL21 (DE3).

[0018] In a fifth aspect, the present invention provides use of the terminal deoxynucleotidyl transferase mutant in enzymatic DNA synthesis.

[0019] Furthermore, in the enzymatic DNA synthesis, deoxyribonucleotides modified with a reversible blocking group at the 3' end are used as substrates, and DNA is synthesized under the catalysis of the terminal deoxynucleotidyl transferase mutant.

[0020] Furthermore, the reversible blocking group is an oxyamino group.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention starts from a truncated terminal deoxynucleotidyl transferase TdT from Zonotrichia albicollis, fixes 52 key residues located in the substrate binding pocket, and obtains 7 terminal deoxynucleotidyl transferase mutant sequences, among which the unfolding temperature T of MP1 / LG-MP5 / LG is m It was more than 10°C higher than the wild type WT / LG. After the obtained mutants were heat-treated at 48°C, 51°C, 56°C, 58°C, and 61°C for 5 minutes, their activity was characterized. Compared with the wild type, the product yield of MP1 / LG-MP5 / LG was higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figures are gel electrophoresis images of different terminal deoxynucleotidyl transferase mutants after incubation at different temperatures; WT / LG represents N-terminally truncated terminal deoxynucleotidyl transferase, N represents primer, and N+1 represents primer extended by 1 base.

[0024] Figure 2 The figure shows the gel electrophoresis of the extension reaction of MP6 / LG and MP7 / LG after incubation at 37°C; wherein N+1 indicates the primer extended by 1 base. DETAILED DESCRIPTION

[0025] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0026] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0028] In the following examples, the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0029] In the following examples, the amino acid sequences of the initial terminal deoxynucleotidyl transferase mutants are shown in SEQ ID NOs. 1 to 7, the nucleotide sequences of the genes encoding the initial terminal deoxynucleotidyl transferase mutants are shown in SEQ ID NOs. 8 to 14, the amino acid sequence of the N-terminally truncated terminal deoxynucleotidyl transferase sZaTdT is shown in SEQ ID NO. 15, and the nucleotide sequence of the N-terminally truncated terminal deoxynucleotidyl transferase sZaTdT is shown in SEQ ID NO. 16.

[0030] SEQ ID NO.1:

[0031] MKISQYSCQRRTSLKNLNQKFVDAFNVMADYYEYLEDKEKAEKYRRAAALLRSLPFKVTKMEDIEGLPNIYEECRKIIKEILEKGYSPEVEKIKNDPYYQAMKEFTSVYGFGVKEAKELYKKGYKTVEEVKADKTLKLSATQKAGLKYYDDLVAPVTREEAEAVHRLITETVHAFVPDAVVEI TGGFRRGKKTTHDVDFLISNPGPVEDPDLLDKVLARLEAEGRLLYAERIPSTYDPDRLPSEKLDAMDHFQKCFTIVKLPLPPPPPAAAAAAAPVPPAPPRGWRAVRVDLTYVPYERYPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLYDRKTGRFLEAASEREIFDHLGLEYIPPEERNA

[0032] SEQ ID NO.2:

[0033] MKISQYSCQRRTSLKDLNKEFTDALKVMAEYYEYKEDKENAEKYRKAAALLRSLPFKVTKLEDIKGLPNISEEVRKIIEEILKNGYSEEVEKIKNDPYYKAMKEFTSVYGFGVKEAKELYAKGYKTVEEVKADKTLKLTEEQKAGLKYYEDLVRPVTREEAEAVYEIISETVHKFVPEAVVEITGGFRRGKKTTHDVDFLISNPGPEEHPDLLQKIIEELKKQGLLLYYELIPSTYDPNRLPSEKLDAMDHFQKCFMIIKLPLPPPPPAAAPEAAPVPPAPPRNYRAVRVDLVYVPKERYPYALLGWTGSRQFVRDLRRYALEERGMLLDNHGLYDRKTGRFLKAESEKEIFDHLGLEYIPPEERNA

[0034] SEQ ID NO.3:

[0035] EVVSQYSCQRRCSLTNLNQKFVDAFDIMAEYYEFKENKKEAEKFRRAAALLRCLPFEVTKMEDIEGLPNIYEEERKIIEEILKNGYSPEVEKIKNDPYFKAMKEFTKIYGFGVKRAKEYYAKGYKTVEEIKADKSLKLSETQKAGLKYYEDLVRPITREEALAVHEIIKSTVHKFRPDAIVEITGGFRRGKETTHDVDFLISNPGPVEDPNLLDKIIEELKEKGILLYYEKIPSTYDPNRLPSTKLDAMDHFQKCFMIIKLYLPPPPPAAAAAAAPVPPAPPQDWRAVRVDLTYVPYSRRPYALLGWTGSRQFVRDLRRYALEERGMILDNHGLYDRKTGKFLEAASEEEIFAHLGLPYIPPEQRNA

[0036] SEQ ID NO.4:

[0037] KKYSQYSCQRRTSLKNLNQKFVDAFEVMARYAEFKGDKETAERYRRAAALLRSLPFEVKSMEDIEGLPNIYPECREIIEEILKYGYSKRVEAIKADPYFKAMSEFVSVYGFGVKRAKELIAKGYTTVEQVKADKTLKLSKTQKAGLKYYEDLVRPVTREEAEVHEIITSTVHKFRPDAVVEVTGGFRRGKETTHDVDFLISNPGPVEDPDLLQKIVDTLKEEGLLLYHEIIPSTYKPDALPSEKLDAMDHFQKCFMIIKLPLPPPPPPAAAAAAPAPPAPPKDWRAVRVDLTLVPAEARPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLFDRKAGRFVAAASEEEIFAALGLPYIPPEERNA

[0038] SEQ ID NO.5:

[0039] MVYSQYSCQRRTSLKDLNKEFTDAFDIMAEYAEFKEDKETAEKYRDAAALLRSLPFKVTKMEDIEGLPNIYPDCREIIEEILKNGYSSRVEKIKNDPYFKAMKEFTKVLGFGVKRAKEYYAKGYTTVEQIKADKSIKLTPEQKAGLKYYEDLVAPVTREEAEAVAKIIRETVHKFVPNAIVEITGGFRRGKETTHDVDFLISNPGPVDDPLKKVVEELKKEGLLLYAKIIESTYDPDRLPSEKEDAMDHFQKCFMIIKLYLPPPPPPAAGPAPAPVPPAPPRGYRAVRVDLTYVPYERYPFALLGWTGSRQFVRDLRRYAREERGMLLDNHRLYDRRTGRFVEAKSEQEIYDALGLEYIPPEDRNA

[0040] SEQ ID NO.6:

[0041] MKISQYSCQRRTSLKNLNQKFVDAFNVMADYYEYLEDKEKAEKYRRAAALLRSLPFKVTKMEDIEGLPNIYEECRKIIKEILEKGYSPEVEKIKNDPYYQAMKEFTSVYGFGVKEAKELYKKGYHTHHHHHHDKTLKLSATQKAGLKYYDDLVAPVTREEAEAVHRLITETVHAFVPDAVVEITGGFRRGKHHHHDVDFLISNPGPVEDPDLLDKVLARLEAEGRLLYAERIPSTYDPDRLPSEKLDAMDHFQKCFTIVKLPLPPPPPAAAAAAAPVPPAPPRGWRAVRVDLTYVPYERYPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLYDRKTGRFLEAASEREIFDHLGLEYIPPEERNA

[0042] SEQ ID NO.7:

[0043] MKISQYSCQRRTSLKNLNQKFVDAFNVMADYYEYLEDKEKAEKYRRAAALLRSLPFKPPPPPDPEGLPNIYEECRKIIKEILEKGYSPEVEKIKNDPYYQAMKEFTSVYGFGVKEAKELYKKGYHTHHHHHHDKTLKLSATQKAGLKYYDDLVAPVTREEAEAVHRLITETVHAFVPDAVVEITGGFRRGKHHHHDVDFLISNPGPVEDPDLLDKVLARLEAEGRLLYAERIPSTYDPDRLPSEKLDAMDHFQKCFTIVKLPLPPPPPAAAAAAAPVPPAPPRGWRAVRVDLTYVPYERYPYALLGWTGSRQFVRDLRRYAREERGMLLDNHGLYDRKTGRFLEAASEREIFDHLGLEYIPPEERNA

[0044] SEQ ID NO.8:

[0045]

[0046] SEQ ID NO.9:

[0047]

[0048] SEQ ID NO.10:

[0049]

[0050] SEQ ID NO.11:

[0051]

[0052] SEQ ID NO.12:

[0053]

[0054] SEQ ID NO.13:

[0055]

[0056] SEQ ID NO.14:

[0057]

[0058] SEQ ID NO.15:

[0059] MKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIFCLEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEIIEEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPSRKVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLEMAEVKDWKAIRVDLVITPFEQYPYALLGWTGSRQFGRDLRRYAAHERKMILDNHGLYDRRKRIFLKAGSEEEIFAHLGLDYVEPWERNA

[0060] SEQ ID NO.16:

[0061]

[0062] Example 1 Construction of mutants

[0063] (1) In this study, we selected the terminal deoxynucleotidyl transferase (ZaTdT) from diphtheria, and used AlphaFold 2 to predict the three-dimensional structure of the N-terminally truncated ZaTdT (sZaTdT, WT) as the input for ProteinMPNN. To preserve the catalytic activity of the terminal deoxynucleotidyl transferase, 52 key residues located in the substrate binding pocket were fixed when designing new sequences using ProteinMPNN. Amino acid residues 4, 5, 8, 9, 10, 114, 184, 185, 186, 187, 188, 189, 190, 191, 195, 196, 198, 235, 243, 245, 249, 250, 251, 252, 253, 254, 255, 256, 289, 291, 303, 304, 305, 306, 307, 308, 309, 310, 311, 315, 318, 319, 330, 331, 332, 337, 355, 356, 357, 365, 366, and 367 of sZaTdT were fixed. ProteinMPNN was used to generate 1000 sequences, from which the sequences of seven initial terminal deoxynucleotidyl transferase mutants with the best global score and low sequence similarity were selected.

[0064] The initial terminal deoxynucleotidyl transferase mutant gene was synthesized by Qingke Bioengineering Co., Ltd. and inserted into the restriction sites BamH I and Hind III of the vector pET28a to obtain a recombinant plasmid.

[0065] (2) Construction of the superimposed R189L / K191G mutant

[0066] Based on the amino acid sequences of the seven initial terminal deoxynucleotidyl transferase mutants, R189L / K191G mutations were performed to obtain the sequences of seven terminal deoxynucleotidyl transferase mutants (named MP1 / LG, MP2 / LG, MP3 / LG, MP4 / LG, MP5 / LG, MP6 / LG, and MP7 / LG). The terminal deoxynucleotidyl transferase mutant genes were synthesized by Qingke Bioengineering Co., Ltd., and appropriate primers were designed for whole-plasmid PCR. The PCR reaction system and primers are shown in Tables 1 and 2.

[0067] Table 1 PCR reaction system

[0068]

[0069]

[0070] Note: N* refers to 1 to 5, indicating different types of mutants.

[0071] Table 2 Primers used for mutant construction

[0072]

[0073] Example 2 Expression and purification of different terminal deoxynucleotidyl transferase mutants

[0074] Plasmids carrying different terminal deoxynucleotidyl transferase mutant genes were transformed into BL21 (DE3) cells, spread onto LB solid medium containing 50 μg / mL kanamycin (Kana), and cultured in a 37°C constant temperature incubator for 12 h; a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL Kana, and cultured at 37°C, 220 rpm for 8-12 h; a 2% inoculum was transferred to 50 mL fresh LB liquid medium also containing 50 μg / mL Kana, and cultured at 37°C, 220 rpm, until the OD 600 When the pH value reached about 0.8, IPTG was added at a final concentration of 1 mM and the culture was induced at 18°C ​​and 220 rpm for 16 h.

[0075] After the culture is completed, the bacterial solution is centrifuged at 4000 rpm and 4°C for 15 minutes, the supernatant is discarded, and the collected bacteria are then resuspended in 50 mM phosphate buffer with a pH of 7.5 and ultrasonically disrupted at 400 W in an ice water bath, with each ultrasonication for 3 seconds and an interval of 7 seconds. After the disruption is completed, the cell disruption solution is centrifuged at 12000 rpm and 4°C for 30 minutes to remove cell debris. The supernatant obtained is the crude enzyme solution, which is purified by filtration with a 0.22 μm filter membrane. Impurities are washed with a washing buffer (50 mM PBS buffer, 50 mM imidazole, 100 mM NaCl, pH 7.5), and the target protein is eluted with an elution buffer (50 mM PBS buffer, 250 mM imidazole, 100 mM NaCl, pH 7.5). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain pure terminal deoxynucleotidyl transferase mutant proteins.

[0076] Example 3 Activity Characterization of Different Terminal Deoxynucleotidyl Transferase Mutants

[0077] The terminal deoxynucleotidyl transferase mutant protein prepared in Example 2 was used to prepare a 0.05 mg / mL enzyme solution. The reaction system was 10 μL, including 1 μM primer, 33 μM 3′-ONH2-dGTP, 0.25 mM CoCl2, 100 mM NaCl and 50 mM phosphate buffer (pH 7.5). The 0.05 mg / mL enzyme solution was reacted at 37°C for 10 min. The reaction was analyzed by 20% urea-polyacrylamide gel electrophoresis. The results are shown in FIG. Figure 1 and Figure 2 shown.

[0078] Example 4 Characterization of the thermal stability of different terminal deoxynucleotidyl transferase mutants

[0079] The terminal deoxynucleotidyl transferase mutant protein prepared in Example 2 was used to prepare a 0.05 mg / mL enzyme solution. The 0.5 mg / mL enzyme solution was incubated at 40°C, 45°C, 48°C, 51°C, 56°C, 58°C, and 61°C for 5 min, respectively, and then placed on ice for later use. The reaction system was 10 μL, including 1 μM primer, 33 μM 3′-ONH2-dGTP, 0.25 mM CoCl2, 100 mM NaCl, and 50 mM phosphate buffer (pH 7.5). 1 μL of the above enzyme solution was added to initiate the reaction. The reaction was incubated at 37°C for 10 min, and the reaction was analyzed by 20% urea-polyacrylamide gel electrophoresis. The results are shown in FIG. Figure 1 Grayscale extraction was performed using SHST analysis to measure the grayscale of the extension product and the grayscale of the unextended band. The yield was calculated using the ratio of the grayscale of the extension product to the sum of the grayscales of the extended and unextended bands in the lane. The results are shown in Table 3.

[0080] Table 3 Product yields of terminal deoxynucleotidyl transferase mutants WT / LG, MP1 / LG-MP5 / LG

[0081]

[0082] Example 5 Unfolding Temperature T of Different Terminal Deoxynucleotidyl Transferase Mutants m Determination

[0083] The unfolding temperature (T) of the mutants was determined using a protein thermal stability analyzer (NanoTemper, USA). m A protein sample with a concentration of 0.5 mg / mL was loaded into a quartz capillary, the power was set to 10%, the scanning rate was 60°C / h, and the scanning range was 20-90°C. m As shown in Table 4.

[0084] Table 4 Unfolding temperature T of different terminal deoxynucleotidyl transferase mutantsm

[0085] mutant <![CDATA[T m / ℃]]> WT / LG 46.75±0.05℃ MP1 / LG 74.30±0.10℃ MP2 / LG 64.05±0.25℃ MP3 / LG 82.05±0.15℃ MP4 / LG 81.50±0.30℃ MP5 / LG 64.50±0.90℃ MP6 / LG 40.50±0.20℃ MP7 / LG 42.15±0.15℃

[0086] As can be seen from Table 4, the different terminal deoxynucleotidyl transferase mutants used in this example have higher unfolding temperatures than the wild-type terminal deoxynucleotidyl transferase. The unfolding temperatures T of MP1 / LG, MP2 / LG, MP3 / LG, MP4 / LG, and MP5 / LG are m More than 10℃ higher than the wild type WT / LG.

Claims

1. A terminal deoxynucleotidyl transferase mutant, characterized in that The terminal deoxynucleotidyl transferase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO. 4 to R189L and K191G mutations.

2. A gene encoding the terminal deoxynucleotidyl transferase mutant according to claim 1.

3. A recombinant vector, characterized in that Contains the coding gene as described in claim 2.

4. A genetically engineered bacterium, characterized in that: The method comprises a host cell comprising the encoding gene according to claim 2 or the recombinant vector according to claim 3.

5. The genetically engineered bacterium according to claim 4, characterized in that The host cell is Escherichia coli.

6. The genetically engineered bacterium according to claim 5, characterized in that The host cell is E. coli BL21 (DE3).

7. Use of the terminal deoxynucleotidyl transferase mutant according to claim 1 in enzymatic DNA synthesis.

8. The use according to claim 7, characterized in that In the enzymatic DNA synthesis, deoxyribonucleotides modified with a reversible blocking group at the 3' end are used as substrates, and DNA is synthesized under the catalysis of the terminal deoxynucleotidyl transferase mutant.

9. The use according to claim 8, characterized in that The reversible blocking group is an oxyamino group.

Images