IMMOBILIZED MULTI-ENZYMATIC HALOGENATION SYSTEM
20200299671 ยท 2020-09-24
Inventors
- Sylvie Garneau-Tsodikova (Lexington, KY)
- Oleg V. Tsodikov (Lexington, KY)
- Shogo Mori (Lexington, KY, US)
- Michael D. Burkart (La Jolla, CA)
- James J. La Clair (La Jolla, CA, US)
Cpc classification
C12N9/0071
CHEMISTRY; METALLURGY
C12N11/02
CHEMISTRY; METALLURGY
International classification
Abstract
A halogenation system, a method of halogenating a substrate, and halogenated compounds are provided. The halogenation system includes PltM immobilized on a solid support. The system may include one or more additional enzymes immobilized on the solid support. The method of halogenating a substrate includes running the substrate and a reaction solution through the halogenation system including PltM immobilized on a solid support. The halogenated compounds include 4,6-diCl-3, 4,6-diCl-8, 2,4-diCl-9, 2,6-diCl-11, 3,5-diCl-15, 4,6-diCl-16, 4,6-diCl-18, 4-Cl-23, and/or 4,6-diBr-3.
Claims
1. A halogenation system comprising: PltM; and a solid support; wherein the PltM is immobilized on the solid support.
2. The system of claim 1, wherein the solid support is a resin.
3. The system of claim 2, wherein the resin is an agarose resin.
4. The system of claim 2, wherein the resin is packed into a spin column.
5. The system of claim 1, further comprising one or more enzymes immobilized on the solid support.
6. The system of claim 5, wherein the one or more enzymes include a flavin adenine dinucleotide (FAD) reductase.
7. The system of claim 6, wherein the FAD reductase includes SsuE.
8. The system of claim 5, wherein the one or more enzymes include a NADPH regenerator.
9. The system of claim 8, wherein the NADPH regenerator includes glucose dehydrogenase (GDH).
10. The system of claim 5, wherein the one or more enzymes include a flavin adenine dinucleotide (FAD) reductase and a NADPH regenerator; and wherein the FAD reductase and the NADPH regenerator are immobilized on the solid support.
11. The system of claim 10, wherein the FAD reductase is SsuE.
12. The system of claim 11, wherein the NADPH regenerator is glucose dehydrogenase (GDH).
13. The system of claim 12, wherein the PltM, SsuE, and GDH are packed into a spin column.
14. A method of halogenating a substrate, the method comprising running a substrate and reaction solution through the halogenation system of claim 1.
15. The method of claim 14, wherein halogenation system further comprises SsuE and glucose dehydrogenase (GDH).
16. The method of claim 14, wherein the substrate is a phenyl compound with one or more electron donating groups.
17. The method of claim 16, wherein the phenyl compound is selected from the group consisting of phenolic derivatives, aniline derivatives, short-acting b2 adrenoreceptor agonists, natural products, and a combination thereof.
18. The method of claim 14, wherein the substrate is mono-halogenated.
19. The method of claim 14, wherein the substrate is di-halogenated.
20. A halogenated compound selected from the group consisting of 4,6-diCl-3, 4,6-diCl-8, 2,4-diCl-9, 2,6-diCl-11, 3,5-diCl-15, 4,6-diCl-16, 4,6-diCl-18, 4-Cl-23, and 4,6-diBr-3.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The presently-disclosed subject matter will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
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[0083] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.
DETAILED DESCRIPTION
[0084] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
[0085] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
[0086] Following long-standing patent law convention, the terms a, an, and the refer to one or more when used in this application, including the claims. Thus, for example, reference to a cell includes a plurality of such cells, and so forth.
[0087] The terms comprising, including, and having are intended to be inclusive and mean that there may be additional elements other than the listed elements.
[0088] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
[0089] As used herein, ranges can be expressed as from about one particular value, and/or to about another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as about that particular value in addition to the value itself. For example, if the value 10 is disclosed, then about 10 is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
[0090] As used herein, the term about, when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, or the like is meant to encompass variations of in some embodiments 50%, in some embodiments 40%, in some embodiments 30%, in some embodiments 20%, in some embodiments 10%, in some embodiments 5%, in some embodiments 1%, in some embodiments 0.5%, and in some embodiments 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
[0091] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
[0092] As used herein, nomenclature for compounds, including organic compounds, can be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, Cahn-Ingold-Prelog rules for stereochemistry can be employed to designate stereochemical priority, ElZ specification, and the like. One of skill in the art can readily ascertain the structure of a compound if given a name, either by systemic reduction of the compound structure using naming conventions, or by commercially available software, such as CHEMDRAW (Cambridgesoft Corporation, U.S.A.).
[0093] As used herein, the terms optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
[0094] As used herein, the term subject can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term patient includes human and veterinary subjects.
[0095] As used herein, the term derivative refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.
[0096] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.
[0097] The presently-disclosed subject matter relates to a halogenation system. In some embodiments, the halogenation system includes a bacterial halogenase. Suitable bacterial halogenases include, but are not limited to, PltM. PltM is encoded in the biosynthetic gene cluster of pyoluteorin, an antifungal compound containing a dichloropyrrole moiety. In some embodiments, PltM halogenates substrates with one or more halides, such as, but not limited to, Cl.sup., Br.sup., I.sup., or a combination thereof. This halogenation of the substrate by PltM may include mono-halogenation or di-halogenation with the same or different halogens. For example, in one embodiment, as illustrated in
TABLE-US-00002 TABLE 2 Main Halogenation substrate Halogenase position PDB codes* Phloroglucinol PltM C2 or C2 6BZN (apo) and C4 6BZA (phloroglucinol and partially bound FAD) 6BZQ (FAD) 6BZZ (FAD-partially bound) 6BZT (L111Y, FAD)
[0098] Although discussed above with regard to chlorination of phloroglucinol, the disclosure is not so limited and includes halogenation of other substrates with the same or different halides. In some embodiments, the substrate includes phenyl compounds with electron donating groups. In one embodiment, such compounds include, but are not limited to, phenolic derivatives (e.g.,
[0099] In some embodiments, the halogenation system includes multiple enzymes. In one embodiment, the system includes PltM and at least one other enzyme. In another embodiment, the at least one other enzyme includes one or more of a NADPH regenerator, such as glucose dehydrogenase (GDH), or a flavin adenine dinucleotide (FAD) reductase, such as SsuE. In some embodiments, the enzymes are immobilized on a solid support. Suitable solid supports include, but are not limited to, resins, such as the agarose resin Affi-Gel 15. For example, in one embodiment, the halogenation system includes PltM, SsuE, and GDH immobilized on agarose resin (Affi-Gel 15). In some embodiments, the immobilized enzymes are packed into a spin column, which may be used as a resin conjugate for halogenation. This protein bound resin provides a high halogenation yield for some compounds, which could not be efficiently halogenated by free enzymes in solution. Additionally or alternatively, the enzyme-resin conjugate may be reused 5-6 times without significant loss of efficiency. Without wishing to be bound by theory, this reusability is believed to be the result of a unique recycling mechanism of FAD provided by the combination of immobilized enzymes.
[0100] Also provided herein are methods of using the halogenation system. In some embodiments, the methods include running a substrate and reaction solution through the halogenation system disclosed herein. Any suitable substrate may be used based upon the one or more enzymes within the halogenation system. Suitable substrates include, but are not limited to, phenolic derivatives (e.g.,
[0101] Also provided herein are halogenated compounds formed with the halogenation system. The compounds include mono- and di-halogenated derivatives of any suitable PltM substrate. In one embodiment, the mono-halogenated derivatives include mono-chlorinated derivatives such as, but not limited to, 4-Cl-23 (
[0102] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the presently-disclosed subject matter. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein.
EXAMPLES
Example 1
[0103] This Example describes the characterization PltM and exploration of its ability to halogenate various compounds.
[0104] Results
[0105] Halide Versatility of PltM
[0106] To explore the halide profile of PltM, halogenation of 1 by PltM with NaF, NaCl, NaBr, and NaI used individually in a reaction mixture was tested first. Chlorinated, brominated, and iodinated, but not fluorinated 1, were identified as products (
TABLE-US-00003 TABLE 3 LC/MS data for Assay 1 and Assay 2 against phloroglucinol (1) Obs. Obs. Obs. Calcd. mass mass mass Retention mass [M H].sup. [M + 2 H].sup. [M + 4 H].sup. time Assay Fig. Substrate Product (Da) (Da) (Da) (Da) (min) # # 1 1 126.0317 125.0244 32.306 Std 1b, S1, S8 F-1 144.0223 1a diF-1 162.0129 1a Cl-1 159.9927 158.9851 160.9821 35.098 1b 1b, S1 diCl-1 193.9537 192.9459 194.9428 196.9399 36.841 1b 1b, S1 Br-1 203.9422 202.9345 204.9324 35.174 1c 1b, S1 diBr-1 283.8527 1c I-1 251.9283 250.9207 35.866 1d 1b, S1 diI-1 377.8250 376.8161 39.309 1d 1b, S1 1 Cl-1 159.9927 158.9887 160.9893 33.454 2a 1c, S2 diCl-1 193.9537 2a Br-1 203.9422 202.9402 204.9382 33.932 2a 1c, S2 diBr-1 283.8527 2a Cl,Br-1 237.9032 2a Cl-1 159.9927 158.9890 160.9853 33.029 2b 1c, S3 diCl-1 193.9537 2b I-1 251.9283 250.9252 34.406 2b 1c, S3 diI-1 377.8250 376.8221 38.055 2b 1c, S3 Cl,I-1 285.8894 2b Br-1 203.9422 202.9412 204.9395 33.202 2c 1c, S4 diBr-1 283.8527 2c I-1 251.9283 250.9296 34.121 2c 1c, S4 diI-1 377.8250 376.8314 37.824 2c 1c, S4 Br,I-1 329.8388 2c Cl-1 159.9927 158.9867 160.9834 32.423 2d 1d, S5 diCl-1 193.9537 192.9484 194.9438 196.9387 34.490 2d 1d, S5 I-1 251.9283 250.9217 33.960 2d 1d, S5 diI-1 377.8250 376.8158 37.678 2d 1d, S5 Cl,I-1 285.8894 284.8811 286.8795 36.027 2d 1d, S5 Br-1 203.9422 202.9403 204.9384 32.647 2e 1d, S6 diBr-1 283.8527 2e I-1 251.9283 250.9285 33.572 2e 1d, S6 diI-1 377.8250 376.8273 37.406 2e Br,I-1 329.8388 2e Note: Although we looked for trihalogenated 1, we did not observe any. All masses were measured in negative mode.
[0107] Substrate Profile of PltM
[0108] Having established the halide versatility of PltM, its substrate profile was investigated next. A set of 20 structurally diverse small molecules was tested first, most, but not all of which were, like 1, phenolic (phenolic derivatives, anilines, nitrobenzene derivative) and included L-Trp (
[0109] PltM catalyzed halogenation of 18 of the 20 compounds tested, exhibiting remarkable substrate versatility for phenolic compounds (
[0110] Since the reaction with compound 11 showed very clear signals of chlorinated and iodinated 11, it was also tested for bromination and fluorination (
TABLE-US-00004 TABLE 4 LC/MS data for all tested substrates. Obs. Obs. Obs. mass mass mass Calcd. [M H].sup./ [M + 2 H].sup./ [M + 4 H].sup./ Retention mass [M + H].sup. [M + 2 + H].sup.+ [M + 4 + H].sup.+ time Assay Fig. Substrate Product (Da) (Da) (Da) (Da) (min) # # 2 2 94.0419 93.0361 33.710 Std. S8, S10 Cl-2 128.0029 126.9964 41.664 1b S10 diCl-2 161.9639 1b I-2 219.9385 1d diI-2 345.8352 1d 3 3 110.0368 109.0305 33.240 Std. S8, S11 Cl-3 143.9978 142.9902 144.9869 36.519 1b S11 diCl-3 177.9588 176.9506 178.9481 180.9499 39.638 1b S11 I-3 235.9334 234.9252 36.242/38.268 1d S11 diI-3 361.8301 360.8203 43.131 1d S11 4 4 126.0317 125.0236 30.894 Std. S8, S12 Cl-4 159.9927 158.9839 160.9824 34.591 1b S12 diCl-4 193.9537 1b I-4 251.9283 250.9216 36.852 1d S12 diI-4 377.8250 1d 5 5 126.0317 125.0238 28.929/34.051 Std. S8, S13 Cl-5 159.9927 1b diCl-5 193.9537 1b I-5 251.9283 250.9216 33.935 1d S13 diI-5 377.8250 1d 6 6 124.0524 123.0513 33.483 Std. S8, S14 Cl-6 158.0135 157.0130 159.0110 40.546 1b S14 diCl-6 191.9745 1b I-6 249.9491 1d diI-6 375.8457 1d 7 7 154.0630 153.0581 38.317 Std. S8, S15 Cl-7 188.0240 187.0184 189.0151 39.581 1b S15 diCl-7 221.9850 1b I-7 279.9596 278.9604 41.04 1d S15 diI-7 405.8563 1d 8 8 143.9978 142.9949 144.9918 34.503 Std. S8, S16 Cl-8 177.9588 176.9571 178.9542 180.9509 37.481 1b S16 I-8 269.8945 268.8947 270.8920 39.385 1b S16 9 9 124.0524 123.0522 34.559 Std. S8, S17 Cl-9 158.0135 157.0140 159.0111 37.561 1b S17 diCl-9 191.9754 190.9761 192.9726 194.9695 40.863 1b S17 I-9 249.9491 248.9543 37.418/39.341 1d S17 diI-9 375.8457 374.8504 42.605 1d S17 10 10 182.0579 181.0577 33.725/33.985 Std. S8, S18 Cl-10 216.0189 215.0194 217.0166 36.019 1b S18 diCl-10 249.9800 1b I-10 307.9546 306.9588 36.538/37.232 1d S18 diI-10 433.8512 1d 11 11 140.0473 139.0396 29.450/29.633 Std. S8, S19 F-11 158.0379 1a diF-11 176.0285 1a Cl-11 174.0084 173.0000 174.9968 32.078 1b S19 diCl-11 207.9694 206.9603 208.9571 210.9538 33.235 1b S19 Br-11 217.9579 216.9563 218.9548 31.923/32.405 1c S19 DiBr-11 295.8684 1c I-11 265.9440 264.9321 33.187/33.529 1d S19 diI-11 391.8406 1d 12 12 138.0317 137.0243 33.265/33.518 Std. S8, S20 Cl-12 171.9927 170.9844 172.9814 36.113 1b S20 diCl-12 205.9537 204.9444 206.9408 208.9364 37.796 1b S20 I-12 263.9283 262.9201 36.208/37.551 1d S20 diI-12 389.8250 1d 13 13 152.0473 151.0391 33.227/33.518 Std. S8, S21 Cl-13 186.0084 184.9992 186.9964 35.651 1b S21 diCl-13 219.9694 1b I-13 277.9440 276.9325 36.198/36.740/36.980 1d S21 diI-13 403.8406 1d 14 14 154.0266 153.0176 33.616 Std. S8, S22 Cl-14 187.9876 1b diCl-14 221.9487 1b I-14 279.9233 278.9143 37.380 1d S22 diI-14 405.8199 1d 15 15 168.0423 167.0416 35.985 Std. S8, S23 Cl-15 202.0033 200.9954 202.9931 98.057 1b S23 diCl-15 235.9643 1b I-15 293.9389 292.9421 39.295 1d S23 diI-15 419.8355 1d 16 16 109.0528 108.0491 31.116 Std. S8, S24 Cl-16 143.0138 142.0117 144.0086 35.795/37.079 1b S24 diCl-16 176.9748 175.9735 177.9707 179.9676 40.487 1b S24 I-16 234.9494 233.9476 37.974/38.929 1d S24 diI-16 360.8460 359.8479 44.288 1d S24 17 17 153.0790 154.0863 33.634 Std. S8, S25 Cl-17 187.0400 188.0466 190.0432 38.621 1b S25 diCl-17 211.0010 1b I-17 278.9756 279.9806 44.046 1d S25 diI-17 404.8723 1d 18 18 108.0687 107.0669 30.101 Std. S8, S26 Cl-18 142.0298 141.0268 143.0239 36.581 1b S26 diCl-18 175.9908 1b I-18 233.9654 1d diI-18 359.8620 1d 19 19 212.0069 211.0327 35.780 Std. S9 Cl-19 245.9680 1b diCl-19 279.9290 1b I-19 337.9036 1d diI-19 463.8002 1d 20 20 204.0899 203.0795 29.860 Std. S9 Cl-20 238.0509 1b diCl-20 272.0119 1b I-20 329.9865 1d diI-20 455.8832 1d 21 21 225.1365 224.1264 27.654 Std. S8, S27 Cl-21 259.0975 1b diCl-21 293.0585 1b I-21 351.0331 350.0230 28.222/28.485 1d S27 diI-21 476.9298 475.9219 29.804 1d S27 22 22 303.1471 304.1543 30.691/34.162 Std. S8, S28 Cl-22 337.1081 1b diCl-22 371.0691 1b I-22 429.0437 430.0494 32.154 1d S28 diI-22 554.9403 1d 23 23 228.0786 227.0794 35.871/36.280/37.368 Std. S8, S29 Cl-23 262.0397 261.0418 263.0390 37.565/38.532 1b S29 diCl-23 296.0007 1b I-23 353.9753 352.9816 38.531 1d S29 diI-23 479.8719 1d 24 24 290.0790 289.0838 31.263/31.372/31.712 Std. S8, S30 Cl-24 324.0401 323.0450 325.0415 33.137 1b S30 diCl-24 358.0011 1b I-24 415.9757 414.9837 33.533/34.204 1d S30 diI-24 541.8723 1d Note: Although we looked for halogenation beyond two sites, we did not observe any for the substrates tested. *All compounds were measured in negative mode except for 3,5-dimethoxyaniline (17) and fenoterol (23)
[0111] Crystal Structure of PltM and its Complex with Phloroglucinol
[0112] In addition to its remarkable halide versatility and a very broad substrate profile for a phenolic halogenase, PltM is at most 15% identical in sequence to other structurally characterized FAD-dependent halogenases, and it contains a unique C-terminal region (residues 390-502) (
[0113] A monomer of PltM (
[0114] The binding site of compound 1 is analogous to that of L-Trp in the crystal structure of RebH and PrnA (
TABLE-US-00005 TABLE 5 X-ray diffraction data collection and structure refinement statistics for apo-PltM and apo-PltM-Hg. PltM PltM-Hg PDB ID 6BZN 6BZI Data collection Space group P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 Number of monomers per asymmetric unit 4 4 Unit cell dimensions a, b, c () 64.2, 157.1, 214.0 63.7, 156.3, 216.1 , , () 90, 90, 90 90, 90, 90 Resolution () 39.88-1.80 (1.83-1.80) 50.0-2.4 (2.44-2.40)
R.sub.merge 0.125 (0.766) 0.164 (0.708) I/I 14.5 (1.7)
11.2 (2.4)
Completeness (%) 99.5 (96.3) 96.3 (92.2) Redundancy 6.7 (6.1) 6.7 (6.5) Structure refinement statistics Resolution () 39.88-1.80 45.0-2.4 Number of unique reflections 189997 78214 R
/R.sub.free 0.161/0.189 0.209/0.256 No. of atoms Protein 15852 15595 Ligand/Ion 67 71 Water 2015 451 B-factors Protein 19.6 28.0 Ligand/Ion 31.4 27.3 Water 31.3 24.6 R.m.s. deviations Bond lengths () 0.02 0.008 Bond angles () 1.71 1.19 Ramachandran plot statistics.sup.b % of residues in favored region 98.1 98.3 % of residues in allowed region 1.9 1.7 % of residues in outlier region 0 0 Ligands/Ions Glycerol (10)
Glycerol (3) Calcium (7) Calcium (4) Mercury (25) Ethylmercury (8) .sup.aNumbers in parentheses indicate the values in the highest-resolution shell. .sup.bIndicates Rampage statistics.
.sup.cNumber of ligands in the asymmetric unit.
indicates data missing or illegible when filed
[0115] Crystal Structures of PltM with FAD Bound in Different States
[0116] PltM represents a type of FAD-dependent enzyme, where FAD dissociates out of its binding site for reduction. To gain structural insight into this enigmatic process, a crystal structure of PltM-FAD complex was determined by soaking the crystals of apo PltM with FAD. Two different crystal forms of PltM-FAD complexes were obtained, where a molecule of FAD was bound to PltM in two different states (
[0117] A short nonconserved loop containing three Ala, a Gly and a Ser (residues 172-178) and the side chain of Gln321 are in two different conformations in these two structures (
[0118] Halogenation Assays in Fermentation Culture
[0119] As a preliminary assessment of potential use of PltM in a fermentation setting, the ability to halogenate phloroglucinol (1) upon addition to the culture of E. coli BL21(DE3) overexpressing PltM was tested. The substrate binding cavity observed in the crystal structures was also validated by testing halogenation by two PltM point mutants of PltM, L111Y and S404Y, in this setting. These two residues (one from the FAD binding fold and one from the C-terminal region) line the substrate binding cavity, and their bulkier substitutions are predicted to block binding of 1 (
[0120] A crystal structure of PltM L111Y was determined, which showed that the overall protein structure is unperturbed and the only effect of the mutation was to obstruct the access to the substrate binding pocket, as predicted (
[0121] Kinetics and Regiospecificity of PltM in Optimized Reactions
[0122] For quantitative analysis of enzyme kinetics and detailed structural characterization of reaction products, as well as for potential future biotechnological use, in vitro enzymatic reaction conditions were extensively optimized and enzymes were coupled to maximize product yield. The critical factors of the optimized conditions were introducing glucose dehydrogenase (GDH) for NADPH regeneration and lowering the concentrations of NADPH and halide salts. This optimization significantly improved reaction yields, resulting in full conversion of several substrates (Table 8). This additional information corroborated the preference for substrates containing electron-withdrawing groups and showed preference of PltM for substrates with 1- and 3-hydroxyl or amino groups.
TABLE-US-00006 TABLE 8 Overall yield of optimized chlorination reactions for different substrates of PltM. % overall conversion.sup.a Substrate (trial 1, 2).sup.b 2 57, 25 3 100, 100 6 1, 4 8 100, 100 9 97, 96 10 3, 2 11 100, 100 12 28, 26 13 5, 3 15 34, 7 16 100, 100 18 100, 100 23 24, 20 .sup.a% overall conversion is the sum of all chlorinated products. .sup.bYields of two independent reactions are reported.
[0123] The halide preference was determined and the kinetics of chlorination and bromination of substrates 3, 11, and 16 was evaluated quantitatively, which showed 100% conversion upon overnight reaction (
TABLE-US-00007 TABLE 9 Kinetic parameters for halogenations of selected substrates. Substrate Halogen Substrate/Product k.sub.cat (min.sup.1) K.sub.m (M) k.sub.cat/K.sub.m (min.sup.1M.sup.1) 3 Cl 3/Cl-3 2.3 0.1.sup.a .sup.(7.6 1.3) 10.sup.2 30 5.sup. Cl-3/diCl-3 0.40 0.01 0.11 0.02 3.5 0.6.sup. Br 3/Br-3 1.9 0.1 0.71 0.2 2.7 0.6.sup. Br-3/diBr-3 0.38 0.01 0.78 0.17 0.49 0.11.sup. 11 Cl 11/Cl-11 1.6 0.1 8.4 0.4 0.19 0.01.sup. Cl-11/diCl-11 0.18 0.01 0.44 0.02 0.41 0.02.sup. Br 11/Br-11 0.24 0.02 (1.1 0.2) 10.sup.3 (2.2 0.2) 10.sup.4 16 Cl 16/Cl-16a.sup.b 0.35 0.01 1.1 0.3 0.31 0.09.sup. 16/Cl-16b 0.22 0.01 1.1 0.3 0.19 0.06.sup. Cl-16a/diCl-16 0.95 0.01 18 5 (5.2 1.4) 10.sup.2 Cl-16b/diCl-16 1.0 0.01 15 9 (7.0 4.4) 10.sup.2 Br 16/Br-16a 0.47 0.04 151 20 (3.1 0.5) 10.sup.3 16/Br-16b 2.2 0.1 151 20 (1.5 0.2) 10.sup.2 Br-16a/diBr-16 0.21 0.02 6.0 1.2 (3.5 0.8) 10.sup.2 Br-16b/diBr-16 2.0 0.1 (1.3 0.1) 10.sup.3 (1.5 0.2) 10.sup.3 .sup.aThe values of all mono-halogenation and dihalogenation rate constants k.sub.cat, 1 and k.sub.cat, 2 respectively, and K.sub.m for mono-halogenation and dihalogenation (defined as (k.sub.d, 1 + k.sub.cat, 1)/k.sub.a, 1 and (k.sub.d, 2 + k.sub.cat, 2)/k.sub.a, 2, respectively) were determined by nonlinear regression using Dynafit, as described in Methods. .sup.bTwo distinct mono-halogenation products of the same reaction are denoted by labels a and b.
[0124] The high yield of chlorination and bromination of these and several other compounds allowed the present inventors to establish the regiospecificity of the halogenation by PltM. However, some substrates or products were insufficiently stable during halogenation reactions precluding their quantitative structural analysis. The structures of the final dichlorinated products of 3, 8, 9, 11, 15, 16, 18, as well as the monochlorinated product of 23 and the dibrominated product of 3 were determined by NMR spectroscopy. The resulting products were 4,6-diCl-3, 4,6-diCl-8, 2,4-diCl-9, 2,6-diCl-11, 3,5-diCl-15, 4,6-diCl-16, 4,6-diCl-18, 4-Cl-23, and 4,6-diBr-3, respectively (
[0125] These results show that for mono- or di-hydroxylated or aminated substrates, PltM halogenates almost exclusively in ortho to these polar groups, but not between them. However, when a methyl or a styrene moiety was found in meta to two hydroxyls, as in compound 9 (which was dichlorinated) and resveratrol (23; which was monochlorinated), respectively, a chlorination event occurred between the two hydroxyls.
[0126] Development of an Immobilized Halogenating System
[0127] The halogenation yield is limited by stability of proteins, with PltM being the limiting factor. To achieve a more efficient and scalable halogenation reaction, the present inventors developed a method where all three proteins were immobilized on agarose resin (Affi-Gel 15), packed into a spin column, and then used as a resin conjugate for halogenation. The halogenation reactions were performed by adding substrate and reagents into the column. This protein bound resin showed a high halogenation yield for some compounds, which could not be efficiently halogenated by free enzymes in solution (
[0128] The remarkable halide versatility for any FAD-dependent halogenase and very broad substrate profile for a phenolic halogenase call for future exploration of PltM as a halogenation tool. The structures discussed herein revealed a unique architecture of this enzyme, and an FAD orientation that may be relevant to the FAD recycling mechanism shared by FAD binding enzymes.
[0129] Methods
[0130] Materials and Instrumentation
[0131] The PltM, SsuE, and PltA (used as a control in this study) proteins were overexpressed and purified based on our previously described protocols. DNA primers for PCR were purchased from Integrated DNA Technologies (IDT; Coralville, Iowa, USA). Restriction enzymes, Phusion DNA polymerase, and T4 DNA ligase were purchased from New England BioLabs (NEB; Ipswich, Mass., USA). All chemicals and buffer components were purchased from Sigma-Aldrich or VWR (Radnor, Pa., USA) and used without any further purification. Size-exclusion chromatography was performed on a fast protein liquid chromatography (FPLC) system BioLogic DuoFlow (Bio-Rad; Hercules, Calif., USA) by using a HiPrep 26/60 S-200 HR column (GE Healthcare, Piscataway, N.J., USA). Liquid chromatography-mass spectrometry (LC-MS) was performed on a Shimadzu high-performance liquid chromatography (HPLC) system equipped with a DGU-20A/3R degasser, LC-20AD binary pumps, a CBM-20A controller, a SIL-20A/HT autosampler (Shimadzu, Kyoto, Japan), and Vydac HPLC DENALI Column (C18, 2504.6 mm, 5cm particle size) from Grace (Columbia, Md., USA) and an AB SCIEX TripleTOF 5600 (AB SCIEX, Redwood City, Calif.) mass spectrometer recording in negative or positive mode between 80 and 600 m/z. HPLC was performed on an Agilent Technologies 1260 Infinity system equipped with a Vydac HPLC DENALI column (C18, 2504.6 mm, 5cm particle size) and an Alltech Econosil HPLC column (C18, 25010 mm, 10cm particle size; Grace) for analytical and semi-preparative experiments, respectively. .sup.1H and .sup.13C NMR spectra were recorded at 400 and 500 (for .sup.1H) as well as 100 MHz (for .sup.13C) on a Varian 400 MHz spectrometer, using deuterated solvents as specified. Chemical shifts (d) are given in parts per million (ppm). Coupling constants (J) are given in Hertz (Hz), and conventional abbreviations used for signal shape are as follows: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets; br s, broad singlet; dt, doublet of triplets.
[0132] Synthesis of Compound 15
[0133] Aluminum chloride (1.3 g, 9.99 mmol) was slowly added to a solution of phloroglucinol (1, 315 mg, 2.50 mmol) in 1:1/1,2-dichloroethane:nitrobenzene (10 mL) at 0 C. After stirring this mixture at this temperature for 10 min under a nitrogen atmosphere, acetyl chloride (0.21 mL, 3.00 mmol) was added. Then the ice bath was removed, and the mixture stirred at 80 C. for 2 h. The reaction progress was monitored by TLC (1:2/EtOAc:Hexanes, R.sub.f 0.35). The reaction mixture was quenched with H.sub.2O (60 mL), extracted with EtOAc (2100 mL), washed with brine (20 mL), and then dried over MgSO.sub.4. The organic layer was removed under reduced pressure and the residue was purified by flash column chromatography (SiO.sub.2, 1:2/EtOAc:Hexanes) to afford the known compound 15.sup.30 (223 mg, 53%) as a yellow solid: .sup.1H NMR (400 MHz, CD.sub.3OD) 5.78 (s, 2H), 2.58 (s, 3H); .sup.13C NMR (100 MHz, (CD.sub.3).sub.2SO) 203.1, 164.9, 164.5, 104.2, 94.1, 31.3.
[0134] PltM Mutagenesis
[0135] PltM mutants K87A, L111Y, and S404Y were constructed by splicing-by-overlap-extension method. The sequences downstream and upstream of the mutation site were amplified first individually from ppltM-pET28a(NHis). For PltM K87A mutant the primer pairs were: 5-CGCCTGCGGGATCgcgCTGGGCTTCAGTTTTG-3 (SEQ ID NO: 1) with 5-CATACTCGAGCTAGACTTTGAGGATGAAACGATTG-3(SEQ ID NO: 2); and 5-CAAAACTGAAGCCCAGcgcGATCCCGCAGGCG-3 (SEQ ID NO: 3) with 5-GCAGCTCTCATATGAATCAGTACGACGTCATTATC-3 (SEQ ID NO: 4). For PltM L111Y mutant the primers were: 5-CTTGTGGCCCCGCCGtatAAGGTGCCGGAAGCC-3 (SEQ ID NO: 5) with SEQ ID NO: 2; and 5-GGCTTCCGGCACCTTataCGGCGGGGCCACAAG-3 (SEQ ID NO: 6) with SEQ ID NO: 4. For PltM S404Y mutant, the primer pairs were: 5-CTGGCTCAGCGGCtatAACCTGGGCAGTGC-3 (SEQ ID NO: 7) with SEQ ID NO: 2; and 5-GCACTGCCCAGGTTataGCCGCTGAGCCAG-3 (SEQ ID NO: 8) with SEQ ID NO: 4. The PCR products of the above primer pairs were used as templates for another round of PCR using primers SEQ ID NO: 2 and SEQ ID NO: 4. The products from the second round of PCR were digested with restriction enzymes NdeI and XhoI and ligated into NdeI/XhoI-linearized pET28a, yielding ppltMK87A-pET28a, ppltML111Y-pET28a, and ppltMS404Y-pET28a. The mutations were verified by DNA sequencing (Eurofins Genomics).
[0136] Preparation of Pgdh-pET28a Overexpression Construct
[0137] The glucose dehydrogenase (gdh) gene was amplified from genomic DNA of Bacillus subtilis subsp. subtilis 168 by PCR with the forward and reverse primers: 5-AGGATGCATATGTATCCGGATTTAAAAGGAAAAG-3 (SEQ ID NO: 9) and 5-CGCTTTCTCGAGTTAACCGCGGCCTGCCTGGAAT-3 (SEQ ID NO: 10), respectively. The PCR product was purified by agarose gel extraction and digested by restriction enzymes NdeI and XhoI, which was subsequently ligated into NdeI/XhoI-linearized pET28a. The resulting plasmid pgdh-pET28a was transformed into a chemically competent E. coli TOP10 strain, and the cloning was verified by sequencing of the purified plasmids.
[0138] Preparation of PltM and Coupled Enzymes for In Vitro Assays
[0139] Open reading frames encoding PltM and FAD reductase SsuE were cloned into E. coli expression vectors as previously reported. For production of PltM, SsuE, and GDH, the expression vectors were transformed into E. coli BL21 (DE3) (ATCC; Manassas, Va.). In each case, a colony was grown overnight at 37 C. with shaking at 200 rpm in LB medium (5 mL) supplemented with 50 g/mL kanamycin. These overnight cultures were inoculated into LB medium (1 L) supplemented with 50 g/mL kanamycin. Cultures were grown (37 C., 200 rpm) until an attenuance at 600 nm of 0.6 was reached. At this time, protein expression was induced by adding isopropyl--
[0140] Preparation of PltM for Crystallography
[0141] Wild-type PltM and PltM L111Y mutant were purified as described above with an additional size-exclusion chromatography step. Wild-type PltM and PltM L111Y eluted from Ni.sup.II resin were loaded onto an S-200 column equilibrated in 40 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM ME. Fractions containing NHis.sub.6-PltM were pooled and concentrated to 40 mg/mL by using an Amicon Ultra-15 Centrifugal Filter Unit with 10 kDa MWCO. Purified PltM proteins were kept on ice for crystallization studies.
[0142] In Vitro Assays of PltM with Various Substrates and Halides
[0143] The halogenation assays were carried out similarly to a recently described procedure. The substrates that have been tested are given in
[0144] To establish if hetero-dihalogenation by PltM could be observed, halogenating competition assays in 1:1 or 10:1 mixtures of two different halide salts were performed (Assay 2). The reactions contained the same components as above except single halide salts were replaced with either a 1:1/NaCl:NaBr (Assay 2a), 1:1/NaCl:NaI (Assay 2b), or 1:1/NaBr:NaI (Assay 2c) mixtures (100 mM of each halide). The reactions were initiated by adding NADPH under N.sub.2. A 1:1/NaCl:NaBr reaction was also performed with 200 mM of each halide to test occurrence of homo-di- or hetero-chlorination/bromination, and 10:1/NaCl:NaI (Assay 2d) and 10:1/NaBr:NaI (Assay 2e) mixtures with 200 mM of NaCl or NaBr and 20 mM of NaI were tested to check whether chlorination or bromination could occur in the presence of iodide and whether iodination can occur with chlorination or bromination to yield a C1,I-substrate or Br,I-substrate. The reactions were incubated and processed as described above in Assay 1.
[0145] Optimized In Vitro PltM Halogenation Assay
[0146] To increase production of halogenated molecules and decrease the amount of NADPH required, the above in vitro assay was optimized by using an additional enzyme, glucose dehydrogenase (GDH). The optimized reaction mixture contained substrate (0.5 mM for chlorination and bromination; 0.25 mM for iodination; prepared from 50 mM stock in DMSO), FAD (5 M), NADPH (5 M), PltM (6 M), SsuE (5 M), GDH (0.5 M), glucose (20 mM), NaX (10 mM for chlorination and bromination; 0.5 mM for iodination), and sodium phosphate (30 mM, pH 7.4), and was incubated at room temperature. The overall yield of halogenation products was determined for reactions run overnight for several substrates (Table 8). Conversion of the substrate to halogenated products was monitored by HPLC at =275-320 nm, where the absorbance of molecules is not affected by halogenation, and quantified as fraction of reaction species (%).
[0147] The time course experiments for kinetic analysis were performed in 100 L reaction mixtures by quenching the reactions at 0, 5, 15, 30, 60, 120, 240, and 360 min (for 3 and 16), and an additional 720 min (for 11) for chlorination and bromination, and at 0, 30, 60, 120, 240, and 480 min (for 3 and 16), or an additional 720 min (for 11) for iodination. The time course experiments were performed in duplicate. Compound 1 was unstable under these optimized conditions, and it was not tested. The in vitro analysis of K87A mutant was performed overnight in 100 L reaction mixture by using the compound 11 as a substrate. Wild-type PltM was used as a positive control, and no enzyme reaction was used as a negative control. In all above reactions, the compounds were extracted with EtOAc (4100 L) and dried under gentle air flow. The products were dissolved in MeOH (30 L for chlorination and bromination; 15 L for iodination) for HPLC analysis.
[0148] The scale-up experiments were performed overnight in 25 mL for compound 23, in 50 mL for compounds 3, 8, 9, 11, 15, and 18, or 100 mL for 16. PltM concentration was 25 M with compounds 15 and 250 M with compound 23. To process the chlorination reaction of compound 23, ice-cold MeOH (50 mL) was added to precipitate the proteins. This mixture was incubated for 2 h at 20 C., and the protein precipitate was removed by centrifugation (40,000g, 30 min, 4 C.). The pellet was washed by ice-cold MeOH (50 mL) and centrifuged down again (40,000g, 15 min, 4 C.). The supernatant was combined in a round bottomed flask, and MeOH was removed by in vacuo. The products were extracted with EtOAc (4 reaction volume) and dried in vacuo. These were dissolved in MeOH (0.5-1 mL) for purification by semi-preparative HPLC.
[0149] Halogenation Assay Using Immobilized Enzymes
[0150] To increase the yield of halogenation reaction and make the enzymes reusable, PltM, SsuE, and GDH, we immobilized these proteins on Affi-Gel 15 resin (Bio-Rad, Hercules, Calif.). To increase the stability of the coupled enzymes, GDH from Bacillus amyloliquefaciens SB5 (GDH-BA) was used in this assay. This enzyme was expressed and purified, as described above, from a pET23a vector (amp.sup.R) containing a synthetic gene encoding this enzyme (NCBI accession # JQ305165) with an NHis.sub.6 tag, purchased from GenScript (Piscataway, N.J.). The enzymes were dialyzed into buffer C, which contains HEPES (50 mM, pH 7.5), ME (2 mM), and glycerol (10%). Suspended Affi-Gel resin (250 pL) was transferred into a QIAquick spin column (Qiagen), and the resin was washed three times with 500 L of H.sub.2O and buffer D (30 mM HEPES, pH 7.5). For each time, the wash solution was removed by centrifugation (400g, for 15-30 s, 4 C.). The washed resin was incubated with SsuE (50 M, 300 L) for 4 h at 4 C. The beads were washed with buffer D twice and subsequently incubated with a mixture of GDH-BA (200 M, 50 L) and PltM (500 M, 250 L) overnight at 4 C. This resin-enzyme conjugate was washed twice with buffer D and preserved in 4 C. in buffer D until needed. For each 250 L resin, 300 L of reaction solution, which contained substrate (0.5 mM), FAD (5 M), NADPH (5 M), glucose (20 mM), NaCl (10 mM), and HEPES (30 mM, pH 7.5), was used. The reaction with resveratrol (23) was performed overnight at room temperature. The reaction solution was collected by centrifugation (400g, every 15-30 s until the solution was removed, 4 C.), and the resin-enzyme conjugate in the column was washed with buffer D (300 L) three times. These solutions were extracted with EtOAc (4300 L) and dried in vacuo. The solid material was dissolved in MeOH (200 L) and analyzed by HPLC (
[0151] Kinetic Analysis of PltM Halogenation
[0152] To determine the halogenation preference, the kinetic parameters were obtained by the global nonlinear regression analysis of all reaction species using DynaFit software for the following halogenation mechanism:
where E, S, P.sub.1, P.sub.2 are enzyme, substrate, mono- and dihalogenated product, respectively.
[0153] Cell-Based Activity Assay of PltM
[0154] E. coli BL21 (DE3) cells were transformed with ppltM-pET28a, ppltMK87A-pET28a, ppltML111Y-pET28a, ppltMS404Y-pET28a, and ppltA-pET28a. The ppltA-pET28a plasmid overexpressing the halogenase PltA whose substrate is pyrrolyl-S-PltL (a peptidyl carrier protein-linked pyrrole) was used as a negative control. Five colonies from each transformant were cultured in 2500 mL of LB medium (for ppltM-pET28a, ppltML111Y-pET28a, and ppltMS404Y-pET28a) and 1500 mL of LB medium (for ppltMK87A-pET28a and ppltA-pET28a) with 50 g/mL kanamycin at 37 C. and 200 rpm until attenuance of 0.2 at 600 nm. The cultures were then moved to 25 C. until attenuance of 0.5. Protein expression was induced by adding 0.2 mM IPTG to all seven flasks, and the cultures were incubated with shaking for 1 h. 12.5 g/mL of compound 1 was added to 1500 mL of LB medium containing ppltM-pET28a, ppltMK87A-pET28a, ppltML111Y-pET28a, ppltMS404Y-pET28a, and ppltA-pET28a. Compound 1 was not added to the three remaining flasks (negative controls). After additional incubation for 20 h, the cells were pelleted at 5,000 g for 10 min, and the supernatant was collected. The supernatant was extracted with EtOAc (3330 mL), which was dried in vacuo. This was then dissolved in MeOH (100 L) prior to addition of H.sub.2O (800 L) followed by centrifugation at 20,000g for 10 min to remove the precipitate. The supernatant was collected and 1 L was diluted into 199 L of MeOH for LC-MS analysis (Table 7).
TABLE-US-00008 TABLE 7 LC/MS data for cell-based assays. Obs. Obs. Obs. Calcd. mass mass mass Retention mass [M H].sup. [M + 2 H].sup. [M + 4 H].sup. time Assay Fig. Enzyme Product (Da) (Da) (Da) (Da) (min) # # PltM WT I 126.0317 125.0243 29.369 Std. 3, S39 Cl-1 159.9927 158.9852 160.9821 32.416 3 3, S39 diCl-1 193.9537 192.9457 194.9430 196.9391 34.565 3 3, S39 PltM K87A I 126.0317 125.0249 29.171 Std. 3, S39 Cl-1 159.9927 3 diCl-1 193.9537 3 PltM L111Y I 126.0317 125.0246 29.372 Std. 3, S39 Cl-1 159.9927 158.9847 160.9824 32.432 3 3, S39 diCl-1 193.9537 192.9453 194.9419 196.9396 34.558 3 3, S39 PltM S404Y I 126.0317 125.0245 29.368 Std. 3, S39 Cl-1 159.9927 158.9847 160.9817 32.393 3 3, S39 diCl-1 193.9537 3 PltA I 126.0317 125.0244 29.456 Std. S39 Cl-1 159.9927 3 diCl-1 193.9537 3 Note: Although we looked for trihalogenated 1, we did not observe any. All masses were measured in negative mode.
[0155] HPLC and LC-MS Analysis of Halogenated Products
[0156] The halogenation reaction products were analyzed by HPLC or LC-MS by injecting 10 L of each sample. The compounds were separated by Reversed-phase HPLC at the flow rate of 0.2 mL/min by using the following program: eluent A=H.sub.2O; eluent B=MeCN; gradient=2% B for 5 min, increase to 100% B over a 30 min period, stay at 100% B for 9 min, decrease to 2% B over a 1 min period, and re-equilibrate the column at 2% B for 30 min.
[0157] For HPLC analysis, the molecules were observed by absorbance at =275 nm as described above. As necessary, the following mass spectrometer was operated in negative and positive modes with the following parameters: For negative mode, mass range, 80-600 m/z in profile mode; temperature, 550 C. and ion spray voltage floating, 4500 V, and for positive mode, mass range, 80-600 m/z in profile mode; temperature, 550 C. and ion spray voltage floating, 4500 V. The presence of each compound was analyzed by extracted ion chromatograph (XIC) with the expected mass 0.05 Da for Assay 1 and Assay 2 and 0.005 Da for Assay 3 (
[0158] The LC-MS was operated by Analyt TF Software (SCIEX, Framingham, Mass.), and the data was analyzed by PeakView (SCIEX). To purify 4 selected scaled-up halogenated products, semi-preparative HPLC was performed by injecting 100 L per injection at 1 mL/min by using the following gradient program with eluent A as H.sub.2O (with 0.1% TFA) (for compounds 3 and 11) or 10 mM ammonium bicarbonate (for 16) and eluent B as MeCN: 2% B for 10 min, increase to 100% B over a 40 min period, stay at 100% B for 5 min, decrease to 2% B over a 1 min period, followed by re-equilibration in 2% B for 9 min. The collected peak fractions were dried under reduced pressure and lyophilized for NMR analysis.
[0159] NMR analysis of products of large-scale halogenation
[0160] The exact position for the various halogenations were determined either by comparison with commercially available standards (4,6-dichlororesorcinol) or by a combination of HMBC and HSQC experiments.
[0161] The analysis of halogenation products is presented as follows:
[0162] Analysis of 4,6-dichlororesorcinol (4,6-diCl-3): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0163] Analysis of 4,6-dibromoresorcinol (4,6-diBr-3): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0164] Analysis of 2,4,6-trichlororesorcinol (4,6-diCl-8): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0165] Analysis of 2,4-dichloro-5-methylresorcinol (2,4-diCl-9): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0166] Analysis of 2,6-dichloro-3,5-dihydroxybenzyl alcohol (2,6-diCl-11): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0167] Analysis of 3,5-dichloro-2,4,6-trihydroxyacetophenone (3,5-diCl-15): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0168] Analysis of 5-amino-2,4-dichlorophenol (2,4-diCl-16): .sup.1H NMR (400 MHz, CD.sub.3OD,
[0169] Analysis of 2,4-dichloro-1,5-diaminobenzene (2,4-diCl-18): .sup.1H NMR (500 MHz, CD.sub.3OD,
[0170] Analysis of 4-chloro-resveratrol (4-Cl-23): .sup.1H NMR (400 MHz, CD.sub.3OD,
[0171] Analysis of resveratrol (23): .sup.1H NMR (400 MHz, CD.sub.3OD,
[0172] Crystallization of PltM
[0173] PltM crystals were obtained by the hanging drop method with drops containing 0.5 L of PltM (40 mg/mL) and 0.5 L of the reservoir solution (0.1 M Tris pH 8, 0.2 M NaCl, 0.1 M CaCl.sub.2) and 12-17% PEG 8000). The drops were equilibrated against 0.5 mL of reservoir solution at 21 C. Long rod-shaped crystals appeared after 1-3 days. The crystals were cryoprotected by a gradual transfer to the solution with the same composition as the reservoir solution, additionally containing 20% glycerol. The crystals were then frozen by a rapid immersion into liquid nitrogen.
[0174] Determination of the Crystal Structure of PltM
[0175] PltM does not contain a sufficient number of Met residues for structure determination by using anomalous signal from selenium atoms in Se-Met PltM. However, PltM contains eight Cys residues, which, if accessible, would react with Hg salts. Hg derivative crystals of PltM were prepared by transferring native crystals from its mother liquor to the reservoir solution containing 1 mM ethyl mercury phosphate (EMP) and incubated overnight. These crystals were cryoprotected similarly to the native crystals. X-ray diffraction data for this and other crystals of PltM were collected at 100 K at the wavelength of 1 at synchrotron beamline 22-ID at the Advanced Photon Source at the Argonne National Laboratory (Argonne, Ill.). All datasets were indexed, integrated and scaled using HKL2000. The structure was determined by the single anomalous dispersion (SAD) method from the EMP derivative data set (using the wavelength of 1.0 ), as follows. A heavy atom search by using direct method-based SHELXD program initially yielded a substructure of 22 Hg atoms in the asymmetric unit. This Hg substructure was used as an input in Autosolve in PHENIX suite to obtain initial phases, which were bootstrapped by difference Fourier analysis to yield the total of 33 Hg atoms and a readily interpretable electron density map, with the figure of merit of 0.71 after density modification. The structure of the Hg-derivatized PltM was then iteratively built by using COOT and refined by using REFMAC5 (Table 5).
[0176] The refined structure contained four monomers of PltM and 33 Hg atoms coordinated to Cys residues per asymmetric unit. A monomer of PltM from this structure was then used as a search model to determine the structure of native PltM by molecular replacement with Phaser in CCP4i suite. The native crystal structure of PltM was then iteratively adjusted and refined by using COOT and REFMAC5, respectively. Table 5 contains data collection and structure refinement statistics for this and other crystal structures in this study. The crystal structure coordinates and structure factor amplitudes for all crystal structures were deposited in the Protein Data Bank under accession codes specified in Tables 5 and 6.
TABLE-US-00009 TABLE 6 X-ray diffraction data collection and structure refinement statistics for PltM-FAD- phloroglucinol, PltM-FAD, PltM L111Y-FAD and PltM-FAD intermediate complexes. PltM-FAD- PltM PltM-FAD phloroglucinol PltM-FAD L111Y-FAD partially bound PDB ID 6BZA 6BZQ 6BZT 6BZZ Data collection Space group P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 P2.sub.12.sub.12.sub.1 Number of monomers per 4 4 4 4 asymmetric unit Unit cell dimensions a, b, c () 64.2, 157.0, 213.7 63.3, 157.7, 213.5 64.0, 157.5, 213.0 63.82, 157.2, 214.0 , , () 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution () 49.71-2.60 (2.64-2.60) 35.00-2.75 (2.81-2.75) 50.00-2.10 (2.14-2.10)
49.55-2.05 (2.09-2.05)
R.sub.merge 0.172 (0.663) 0.15 (0.82) 0.197 (0.989) 0.175 (0.805) I/I 14.0 (2.2)
12.0 (2.0)
10.7 (1.9)
18.1 (3.0)
Completeness (%) 98.9 (99.5) 96.0 (97.5) 94.7 (92.6) 98.2 (93.9) Redundancy 6.0 (6.0) 4.6 (4.6) 5.4 (4.3) 7.5 (7.1) Structure refinement statistics Resolution () 40.00-2.60 35.00-2.75 35.0-2.10 40.00-2.05 Number of unique reflections 62405 54483 113768 125848
/
0.203/0.253 0.230/0.261 0.207/0.244 0.219/0.245 No. of atoms Protein 15796 15796 15899 15801 Ligand/Ion 79 218 228 76 Water 104 164 943 455 B-factors Protein 47.1 35.4 24.9 21.7 Ligand/Ion 70.7 67.3 36.0 33.6 Water 35.5 25.6 27.7 20.5 R.m.s. deviations Bond lengths () 0.007 0.007 0.007 0.007 Bond angles () 1.17 1.095 1.219 1.207 Ramachandran plot statistics
% of residues in favored region 97.8 98.0 98.0 98.4 % of residues in allowed region 2.2 2.0 2.0 1.6 % of residues in outlier region 0 0 0 0 Ligand/Ions phloroglucinol (3) FAD (4) FAD (4) FAD (4) FAD (2) Chloride (4) Chloride (5) Calcium (4) Chloride (2) Bromide (2) Bromide (9) Calcium (2) .sup.aNumbers in parentheses indicate the values in the highest-resolution shell. .sup.bIndicates Rampage statistics.
.sup.cNumber of ligands in the asymmetric unit.
indicates data missing or illegible when filed
[0177] Structure Determination for the PltM-FAD Intermediate
[0178] PltM crystals were soaked in the reservoir solution used to obtained native PltM crystals, with additional 0.5 mM of FAD. The crystals were then gradually transferred to the reservoir solution with 20% v/v PEG 400 and 0.5 mM FAD, prior to quick immersion in liquid nitrogen. The diffraction data were collected and processed as described above. Rigid body refinement followed by restrained refinement were performed starting from the structure of apo PltM. FAD was readily discernable in the omit F.sub.o-F.sub.c map. Refinement and model building was carried out as described above.
[0179] Structure Determination for the Holo PltM-FAD Complex
[0180] Wild-type PltM and the L111Y mutant (each at 40 mg/mL) were crystallized by using the reservoir solution composed of 0.1 M Tris pH 8, 0.2 M NaBr, 0.1 M CaCl.sub.2) and 14% PEG 8000 (10% PEG 8000 in case of the PltM L111Y mutant). The crystals were gradually transferred to the cryoprotectant solution (0.1 M Tris pH 8, 0.2 M NaBr, 1 mM FAD, 16% PEG 8000 (14% PEG 8000 for the PltM L111Y mutant), 20% PEG 400 and 1 mM FAD) and incubated overnight. Prior to rapid freezing via liquid nitrogen, crystals were briefly transferred to the cryoprotectant solution containing additionally 0.2 M sodium dithionite. The crystal structures were determined by a procedure analogous to that described above.
[0181] Structure Determination for PltM-FAD-Phloroglucinol Complex
[0182] Native crystals of PltM were transferred to reservoir solution with 0.5 mM FAD either without or with 1 mM of phloroglucinol for 10 min, then to the cryoprotectant with the same composition, additionally containing 20% v/v PEG 400. After an overnight incubation, the crystals were rapidly frozen in liquid nitrogen. Compounds 1, 2, 3, 8, 21, 23 and 24 were tested. Data collection, processing, and the structure determination were carried out as described above. FAD was clearly discernable in the omit F.sub.o-F.sub.c electron density map. Out of all substrates tested, only compound 1 (phloroglucinol) yielded omit F.sub.o-F.sub.c electron density. Phloroglucinol was built into a very strong and featureful polder omit mF.sub.o-DF.sub.c electron density in three out of four substrate binding sites in the asymmetric unit (
[0183] Data Availability
[0184] The crystal structure coordinates and structure factor amplitudes for all crystal structures were deposited in the Protein Data Bank under accession codes 6BZN, 6BZI, 6BZA, 6BZQ, 6BZT and 6BZZ, as described in Tables 5 and 6. NMR spectra, LC-MS, and other chromatographic data are included in the raw format herein.
[0185] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:
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[0243] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.