Directed evolution of a regioselective halogenase for increased thermostability

Abstract

Compounds and methods are providing involving RebH variants with improved properties. directed evolution based on random mutagenesis was employed to generate a series of RebH variants. RebH variants with improved thermostability and increased activity at elevated temperatures were generated.

Claims

1. An isolated RebH variant polypeptide of SEQ ID NO: 1 comprising at least one amino acid substitution, wherein the at least one amino acid substitution results in improved halogenating activity; and wherein the at least one amino acid substitution is selected from the group consisting of S2P, I52T, A58V, M71V, M71T, M71A, M71C, N75K, E96V, D101G, S110P, S110L, F111S, G112S, G112D, L113D, L113N, L114P, S130L, K145M, K145R, N166S, F171I, K187R, D203A, D203G, T213A, V225I, K237E, V256I, T258A, D264G, T283A, L289P, F312L, T322I, T348A, L380F, T394M, F396Y, F396L, R400C, T413A, E423D, A442V, S448P, L453P, F458S, F458L, E461G, F465C, F465L, N467T, N470S, A476T, A476V, V481A, Q494R, T496R, T496A, G504S, and R509Q, wherein the RebH variant polypeptide is at least 85% identical to SEQ ID NO:1.

2. The RebH variant of claim 1 comprising the amino acid substitutions S2P, M71V, K145M, N467T, N470S, and G112S.

3. The RebH variant polypeptide of claim 1, wherein the polypeptide halogenates an aromatic substrate.

4. The RebH variant polypeptide of claim 1, wherein the polypeptide halogenates a substrate regioselectively.

5. The RebH variant polypeptide of claim 1, wherein the polypeptide displays improved thermostability over wild-type RebH.

6. The RebH variant polypeptide of claim 1, wherein the polypeptide displays increased halogenating activity at an elevated temperature.

7. The RebH variant polypeptide of claim 1, wherein the polypeptide halogenates the wild-type RebH native substrate tryptophan.

8. The RebH variant polypeptide of claim 1, wherein the polypeptide halogenates non-native substrates.

9. The RebH variant polypeptide of claim 1, wherein the polypeptide halogenates a substrate and wherein the substrate comprises a molecule selected from the group consisting of indole, tryptoline, 2-methyltryptamine, eleagnine, pinoline, tetrahydroharmine, debromodesformylflustrabromine, yohimbine, evodiamine, pindolol, carazolol, and carvedilol.

10. The RebH variant polypeptide of claim 1, wherein the polypeptide halogenates using a halogen selected from the group consisting of fluoride, chloride, bromide and iodide.

11. The RebH variant polypeptide of claim 1, wherein the polypeptide displays a prolonged catalyst lifetime.

12. The RebH variant polypeptide of claim 1, wherein the polypeptide displays an increased tolerance to proteolysis.

13. The RebH variant polypeptide of claim 1, wherein the polypeptide displays an increased tolerance to organic solvents.

14. The RebH variant polypeptide of claim 1, wherein the RebH variant polypeptide halogenates in the absence of a harsh chemical oxidant.

15. A RebH variant polypeptide comprising at least one amino acid substitution at position 2, 52, 58, 71, 75, 96, 101, 110, 111, 112, 113, 114, 130, 145, 166, 171, 187, 203, 213, 225, 237, 256, 258, 264, 283, 289, 312, 322, 348, 380, 394, 396, 400, 413, 423, 448, 453, 455, 458, 461, 465, 467, 470, 476, 481, 494, 496, 504 and/or 509 in SEQ ID NO:1, wherein the RebH variant polypeptide is at least 85% identical to SEQ ID NO:1.

16. The isolated RebH variant polypeptide of claim 1, wherein the variant comprises at least a S2P substitution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIGS. 1A-B. Residual activity following incubation at 49 C. for 2 hr. Tryptophan halogenation reactions were performed on tryptophan with 2% (FIG. 1A) and 0.5% (FIG. 1B) enzyme loading. The best variant (designated 1-PVM) from the first generation library contained three mutations: S2P, M71V, and K145M (FIG. 1A). The 1-PVM mutant was used as the parent for the second-generation random mutagenesis library. Variant 4G6 was identified as having 2.5-fold the activity of the parent and harbored the additional amino acid mutations E423D and E461G as well as a silent nucleotide mutation. The third-generation random mutagenesis library used 4G6 as the template. The three best-performing variants from the third round of screening each contained single amino acid mutations. Following recombination, the two best variants were identified as 3-LR and 3-LSR, which possess the additional mutations S130L and Q494R (3-RL) and S130L, N166S, and Q494R (3-LSR) (FIG. 1B).

(3) FIG. 2. Unfolding transitions from thermal denaturation monitored using CD at 222 nm. The melting temperatures of the best mutants identified throughout the rounds of genetic diversification, screening, and recombination were analyzed to probe the relationship between residual activity and thermostability. Melting temperature measurements were conducted in 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10% glycerol, with a protein concentration of 20 M. Thermal denaturation was irreversible and monitored by circular dichroism spectroscopy using an AVIV 202 CD Spectrometer with Peltier temperature controller. Unfolding was monitored at 222 nm in 2 C. increments from 20-90 C. with 2 min equilibration at each temperature. The midpoint of the denaturation curve was determined with SigmaPlot (Systat Software, San Jose, Calif.) after fitting to a 4-parameter sigmoid. Wild-type RebH has a melting temperature of 52.4 C., and that of the most thermostable variant, 3-LSR, is 70.0 C. The 18 C. increase in T.sub.m indicates significant improvement in enzyme stability and is approximately equal to the difference between enzymes of mesophiles and those of thermophiles.

(4) FIG. 3. Activity-temperature profiles of RebH enzymes. To determine if improved thermostability enables reactions at higher temperatures, activity-temperature profiles of RebH variants were constructed. Activity-temperature profiles were constructed using 0.4% purified enzyme with 75 L reactions in 1.5-mL microcentrifuge tubes. Reactions were run in a buffer of 20 mM HEPES (pH 7.4), 6.7% glycerol, and 100 mM NaCl, with 0.5 mM L-tryptophan, 20 mM DTT, and 100 M FAD. Reactions were run overnight at temperatures ranging from 21-45 C. and processed the following day. With the accumulation of beneficial mutations, the optimum temperature (T.sub.opt) increased by at least 5 C., from between 30 and 35 C. for wild-type RebH to 40 C. for 3-LR. Mutant 3-LR was able to produce 100% more 7-chlorotryptophan than wild-type RebH when each acted at their respective T.sub.opt.

(5) FIG. 4. Local environment of the K145M mutation. Overlay of wild-type RebH (grey backbone and cyan side-chain carbon atoms and blue side-chain nitrogen atoms) and 3-LSR (light blue backbone and yellow side-chain carbon atoms, blue nitrogen atoms, and green sulfur atom). Mutation K145M is located near the surface of the protein and in the area of two arginine residues. Without wishing to be bound by theory, it is thought that wild-type RebH increases the density of positive charge in the area with lysine, and 3-LSR might be stabilized by reducing this density by substituting a methionine at this position and the side chain of methionine adopts a conformation that increases its packing with neighboring residues, which might enhance thermostability.

(6) FIGS. 5A-E. Results (conversion %) of RebH variants tested for their ability to chlorinate tryptoline at different catalyst loads.

(7) FIGS. 6A-E. Thermostability results of wild type RebH and RebH variants. Thermostability analyses were performed at different temperatures and catalyst loads for a set time period. The results are given as percent conversion of tryptophan to 7-chlorotryptophan.

(8) FIGS. 7A-B. Thermostability results of wild type RebH and RebH variants. Thermostability analyses were performed at different temperatures and catalyst loads for a set time period. The results are given as percent conversion of tryptophan to 7-chlorotryptophan.

(9) FIG. 8. General reaction scheme for preparative RebH mutant-catalyzed halogenation reactions and synthesized compounds. Cofactor regen system consisted of 0.5 mol % MBP-RebF and 50 U mL-1 glucose dehydrogenase. [a] Yields of isolated products and HPLC conversions are provided in parentheses. [b] In addition to the major product shown, approximately 10% of the 6-chlorinated compound was observed as well. [c] Only HPLC conversions are shown. Chlorination substrates include: 2=tryptoline, 5=eleagnine, 6=pinoline, 7=tetrahydroharmine, 3=debromodesformylflustrabromine, 8=yohimbine, 4=evodiamine, 9=pindolol, 10=carazolol, and 11=carvedilol.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(10) The present embodiments provide compositions for the halogenation of arenes under mild conditions (aqueous solution, pH 6-8). Various features and advantageous details are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the invention, are given by way of illustration only, and not by way of limitation. Various substitutions, modifications, additions, and/or rearrangements will become apparent to those of ordinary skill in the art from this disclosure.

(11) In the following description, numerous specific details are provided to provide a thorough understanding of the disclosed embodiments. One of ordinary skill in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

(12) Biosynthesis offers an appealing alternative to the harsh chemical conditions required to halogenate aryl groups. RebH is a flavin-dependent halogenase which halogenates arenes by employing halide salts and air as the halogen source and terminal oxidant, respectively.

(13) A new method for selective arene halogenation using the flavin-dependent halogenase RebH employs halide salts and air as the halogen source and terminal oxidant, respectively. Improved expression protocols for RebH and its cognate reductase, RebF, enable halogenation of a range of substituted indoles and naphthalenes. While the scope, selectivity, and mild reaction conditions employed highlight the synthetic utility of enzymatic halogenation, the low catalytic efficiency of RebH (the maximum k.sub.cat observed was 1.1 min.sup.1 on the native substrate, tryptophan) clearly hinders its practicality.

(14) Over the course of preparative-scale bioconversions, extensive RebH precipitation was observed, which suggests that significant improvements in product yield might be possible by increasing the stability of this enzyme. Improving enzyme thermostability has multiple benefits, including prolonging catalyst lifetime, increasing enzyme tolerance to stresses such as proteolysis or organic solvents, and enabling reactions to be conducted at higher temperatures, which increases reaction rate and overall process efficiency. Stabilized RebH variants offer improved tolerance towards subsequent mutations aimed at altering other properties, such as substrate scope and specific activity, since mutations are generally destabilizing.

(15) Directed evolution was employed to increase the thermostability of RebH without decreasing its activity. Three rounds of error-prone PCR and high-throughput screening combined with the recombination of stabilizing mutations yielded RebH variants with higher melting temperatures and increased optimum temperatures for activity. The crystal structure of the most thermostable mutant was solved and compared with the wild-type RebH structure in an effort to gain insight into the molecular basis for thermostability.

(16) Stability is an important property of all enzymes, particularly those exposed to the harsh reactions conditions encountered in industrial processes or subjected to laboratory evolution experiments. Proteins use a variety of strategies for stabilization, and comparisons of homologous proteins from mesophiles and thermophiles has not yielded a unifying set of rules for thermostabilization. Increasing the number of hydrogen bonds, improving packing, decreasing surface to volume ratio, increasing the stability of -helices, increasing the number of ionic interactions, and increasing the hydrophobic interactions in the protein core are all examples of mechanisms exploited for improving thermal stability (Petsko, 2001). No single factor seems to dominate, but many small contributions add to create a thermostable protein.

(17) Given the myriad factors and combinations of factors responsible for thermostabilization, predicting beneficial mutations is challenging. Directed evolution based on random mutagenesis was employed to generate a thermostabilized halogenase. The present study improved the thermostability of the tryptophan halogenase RebH, for which there are no known homologues in thermophiles, while also increasing activity at elevated temperatures.

(18) Three rounds of error-prone PCR, recombination, and screening resulted in variant 3-LRS with a T.sub.m 18 C. higher than that of wild type, and variant 3-LR with a T.sub.opt over 5 C. higher than wild type. Different mutants had the highest T.sub.m and T.sub.opt values, which indicates that thermostability and thermoactivity were not coupled strictly. Without wishing to be bound by theory, one hypothesis that might account for this difference is that increased rigidity helps stability but hinders activity. Examining the crystal structure of the most thermostable mutant, 3-LRS, yielded insights into the possible molecular mechanisms of stabilization. Variant 3-LRS, in comparison to wild-type RebH, modifies the charge distribution on the protein surface by removing a lysine from an already positively charged area and introducing an arginine in the place of a neutral glutamine, and increases the stability of the N-terminus with a Ser-to-Pro mutation.

(19) RebH has been engineered for increased thermostability and activity at elevated temperatures, which addresses immediate concerns regarding catalyst efficiency. This work also establishes a robust protocol for further optimization of RebH.

(20) Variants of RebH obtained using the directed evolution procedure outlined herein could be used to address several significant synthetic challenges, including selective and efficient electrophilic arene halogenation (e.g., XBr, Cl). Panels of RebH variants with activity on model substrates can be used to rapidly identify active and selective enzymes for a wide range of small molecule substrates, such as drug candidates or natural products. These initial hits can be rapidly optimized using directed evolution, which allows for systematic late-stage halogenation of biologically active molecules to improve the activity of these compounds. Evolved FDH variants can also be used to catalyze non-natural oxidative halogenation reactions, including olefin halogenation, halocyclization, and iodination, that have proven challenging using small molecule catalysts.

EXAMPLES

(21) Materials and Methods

(22) The following examples are included to demonstrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

(23) Library Construction, Expression, and ScreeningAll genes encoding RebH were cloned into pET-28a between the NdeI and HindIII digestion sites. Mutant libraries were constructed by error-prone PCR, using Taq polymerase with 150 M MnCl.sub.2 (round 1) or 100 M MnCl.sub.2 (rounds 2 and 3). PCR was performed in a volume of 50 L with conditions of 95 C. 30 s, (95 C. 30 s, 55 C. 30 s, 72 C. 90 s) for 20 cycles, 72 C. 10 min. Beneficial mutations were recombined via overlap extension (Heckman and Pease, 2007) with PCR conditions of 98 C. 30 s, (98 C. 10 s, 72 C. 50 s) for 35 cycles, 72 C. 10 min. Plasmids were transformed by electroporation into E. coli containing the chaperone pGro7. Library colonies were picked using an automated colony picker (Norgren Systems) and arrayed in 1-ml 96-well plates containing 300 L LB with 50 g/mL kanamycin and 20 g/mL chloramphenicol. Cells were grown overnight at 37 C., 250 rpm, and 50-100 L of overnight culture was used to inoculate 1 mL TB media (with 50 g/mL kanamycin and 20 g/mL chloramphenicol) in 2-mL 96-well plates. Following growth at 37 C., 250 rpm, to an OD.sub.600=0.9-1, enzyme expression was induced with IPTG and arabinose to final concentrations of 10 M and 0.2 mg/mL, respectively. Protein expression continued for 20 h at 30 C., 250 rpm, after which cultures were harvested by centrifugation and stored at 80 C. until use.

(24) Cell pellets were thawed and suspended in 100 L 25 mM HEPES (pH 7.4) with 0.75 mg/mL lysozyme. After incubation at 37 C., 250 rpm, cells were flash frozen in liquid nitrogen and thawed in a 37 C. water bath. Ten microliters of DNaseI at 1 mg/mL were added and the cells incubated at 37 C., 250 rpm, for 15 min. After centrifugation, 50 L of supernatant were transferred to a microtiter plate for screening.

(25) Libraries were sealed (AeraSeal, Research Products International), incubated at 42 C. for 2 h (round 1), 51 C. for 2 h (round 2), or 54 C. for 3 h (round 3) and then immediately cooled in an ice water bath. Similar to what has been described previously (Payne, Andorfer and Lewis, 2013), tryptophan halogenation reactions of 75 L total volume in 25 mM HEPES (pH 7.4) consisted of: 50 L lysate, 0.5 mM L-tryptophan, 10 mM NaCl, 100 M NAD, 100 M FAD, 20 mM glucose, 2.5 M RebF (reductase), and 50 U/mL glucose dehydrogenase. Reactions were mixed, the plates sealed, and left overnight on the benchtop. Reactions were quenched with an equal volume of methanol and centrifuged, and the supernatant was filtered and analyzed for 7-chlorotryptophan production via HPLC.

(26) Enzyme Purification and Residual Activity DeterminationEnzyme expression and purification procedures were adapted from a previous report (Payne, Andorfer and Lewis, 2013). An overnight starter culture was used to inoculate 50 mL TB media (with 50 g/mL kanamycin and 20 g/mL chloramphenicol). Following growth at 37 C., 250 rpm, until OD.sub.600=0.6-0.8, enzyme expression was induced with IPTG and arabinose to final concentrations of 100 M and 2 mg/mL, respectively. Protein expression continued for 20 h at 30 C., 250 rpm, after which cultures were harvested by centrifugation and stored at 80 C. until use. Cell pellets were thawed, suspended in 15 mL 20 mM HEPES (pH 7.4), 150 mM NaCl, and lysed by sonication. After clarification by centrifugation, halogenases were purified by Ni-NTA affinity chromatography and exchanged into a buffer of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10% glycerol. For crystallography, mutant RebH was further purified by gel filtration chromatography using a HiLoad 16/600 Superdex 200 column (GE Healthcare Life Sciences) into a buffer of 20 mM HEPES (pH 7.4). Protein concentration was determined using A.sub.280 and extinction coefficients calculated based on amino acid composition.

(27) The residual activity was determined following incubation of 50 L of pure protein at 49 C. for 2 h in 1.5-mL microcentrifuge tubes. Tryptophan halogenation reactions consisted of the same reagents described above with the following exceptions: pure protein was substituted for lysate, and the buffer was 20 mM HEPES (pH 7.4), 6.7% glycerol, and 100 mM NaCl. Reactions were run overnight on the benchtop and processed the following day as above.

(28) T.sub.m and T.sub.opt analysesMelting temperature measurements were conducted in 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10% glycerol, with a protein concentration of 20 M. Thermal denaturation was irreversible and monitored by circular dichroism spectroscopy using an AVIV 202 CD Spectrometer with Peltier temperature controller. Unfolding was monitored at 222 nm in 2 C. increments from 20-90 C. with 2 min equilibration at each temperature. The midpoint of the denaturation curve was determined with SigmaPlot (Systat Software, San Jose, Calif.) after fitting to a 4-parameter sigmoid.

(29) Activity-temperature profiles were constructed using purified enzyme with 75 L reactions in 1.5-mL microcentrifuge tubes. Reactions were run in a buffer of 20 mM HEPES (pH 7.4), 6.7% glycerol, and 100 mM NaCl, with 0.5 mM L-tryptophan, 20 mM DTT, and 100 M FAD. Reactions were run overnight at temperatures ranging from 21-45 C. and processed the following day as above.

(30) Crystallization and structure determinationPurified protein was concentrated to 11 mg/mL, and crystals were grown at 20 C. using the hanging drop vapor diffusion method with a reservoir solution of 1.4 M Na/K phosphate buffer (pH 6.8). Rod-like crystals grew in 2-3 weeks and were flash frozen in liquid nitrogen following cryoprotection with the reservoir solution supplemented with 16% glycerol. Data were collected at NE-CAT beamline 24-ID-E at the Advanced Photon Source at Argonne National Laboratory, and processed using HKL2000 (Otwinowski and Minor, 1997). Phases were determined via molecular replacement using Phaser (McCoy, 2007) and wild-type RebH (PDB ID 2OAM) as the search model. Manual model building was performed in Coot (Emsley and Cowtan, 2004), and the structure was refined with PHENIX (Adams, 2010). Figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrdinger, LLC).

(31) Results

(32) Directed evolution for thermostable RebH mutantsThe thermostability of RebH was increased by random mutagenesis and screening followed by recombination of the beneficial mutations. In order to improve thermostability without losing catalytic activity, the screen involved incubating libraries of RebH mutants at elevated temperature and then testing for residual activity. Error-prone PCR was used to generate a library of RebH variants with an average of 2 residue mutations/sequence. The library was expressed in E. coli in 96-well expression plates, the cells lysed, and the supernatant transferred to microtiter plates for heat treatment. Following tryptophan halogenation reactions, residual activity was determined by HPLC analysis.

(33) The first-generation mutant library was constructed using wild-type RebH as the parent, and 1,365 colonies were screened. Mutants with twice the activity of wild type were identified and their improved activities confirmed following purification. The screen emphasizes catalytic activity following heat treatment, therefore the correlation between heat treatment and thermostability was investigated. To test this, the melting temperature of an improved mutant with a single amino acid mutation, S2P, was analyzed by circular dichroism (CD) spectroscopy. The S2P mutant has a T.sub.m 2 C. higher than that of wild-type RebH, indicative of increased stability. The six mutations identified in improved variants from the first round were recombined using overlap extension PCR, and the best variant (designated 1-PVM) from this library contained three mutations: S2P, M71V, and K145M (Table 1, FIG. 1A).

(34) TABLE-US-00001 TABLE 1 Overlap Extension PCR- Improved Thermostability Recombined Variants From First Variants From First Round Round Variants S2P M71V T213A K145M S2P D203A D203A S2P K145M F396Y S2P F396Y T213A S2P M71V M71V S2P T213A S2P M71V K145M (1-PVM)

(35) The 1-PVM mutant was used as the parent for the second-generation random mutagenesis library. Of the 1,008 colonies screened, variant 4G6 was identified as having 2.5-fold the activity of the parent and harbored the additional amino acid mutations E423D and E461G as well as a silent nucleotide mutation. The third-generation random mutagenesis library used 4G6 as the template and contained another 1,008 colonies. The three best-performing variants from the third round of screening each contained single amino acid mutations. Following recombination, the two best variants were identified as 3-LR and 3-LSR, which possess the additional mutations S130L and Q494R (3-RL) and S130L, N166S, and Q494R (3-LSR) (Table 2, FIG. 1B).

(36) TABLE-US-00002 TABLE 2 Improved Thermostability Variants Improved Thermostability Variants From Second Round From Third Round S2P M71V K145M S2P M71V K145M E423D E461G T413A Q494R T394M S2P M71V K145M S2P M71V K145M E423D E461G Q494R E423D E461G (4-G6) S2P M71V K145M S2P M71V K145M E423D E461G K237E D264G S2P M71V K145M E423D E461G S130L S2P M71V K145M E423D E461G T496R S2P M71V K145M E423D E461G G504S S2P M71V K145M E461G T258A L289P S2P M71V K145M E423D E461G N166S S2P M71V K145M E423D E461G Q494R S130L N166S (3-LR) S2P M71V K145M E423D E461G Q494R N166S S2P M71V K145M E423D E461G Q494R S130L N166S (3-LSR) S2P M71V K145M E423D E461G S130L N166S

(37) Characterization of evolved RebH mutantsThe melting temperatures of the best mutants identified throughout the rounds of genetic diversification, screening, and recombination were analyzed to probe the relationship between residual activity and thermostability (FIG. 2). Wild-type RebH has a melting temperature of 52.4 C., and that of the most thermostable variant, 3-LSR, is 70.0 C. The 18 C. increase in T.sub.m indicates significant improvement in enzyme stability and is approximately equal to the difference between enzymes of mesophiles and those of thermophiles.

(38) TABLE-US-00003 TABLE 3 Melting Temperatures ( C.) of WT RebH and Best Variants wt 52.4 S2P M71V K145M E423D E461G (4-G6) 59.9 S2P M71V K145M E423D E461G Q494R S130L (3-LR) 65.6 S2P M71V K145M E423D E461G S130L N166S 67.8 S2P M71V K145M E423D E461G Q494R S130L N166S (3-LSR) 70

(39) To determine if improved thermostability enables reactions at higher temperatures, activity-temperature profiles of RebH variants were constructed (FIG. 3). With the accumulation of beneficial mutations, the optimum temperature (T.sub.opt) increased by at least 5 C., from between 30 and 35 C. for wild-type RebH to 40 C. for 3-LR. Mutant 3-LR was able to produce 100% more 7-chlorotryptophan than wild-type RebH when each acted at their respective T.sub.opt.

(40) RebH has been shown to halogenate a variety of non-native substrates (Payne, Andorfer and Lewis, 2013; Vaillancourt, et al., 2006; Blasiak and Drennan, 2009; Butler and Sandy, 2009; Anderson and Chapman, 2006). The ability of thermostable variants to halogenate the native substrate L-tryptophan as well as non-native substrates tryptamine and tryptoline was investigated. Several variants displayed chlorinating activity towards L-tryptophan non-native substrates tryptamine and tryptoline (Tables 4 and 5). Through methods analogous to the directed evolution of RebH for increased thermostability, further mutations were added to mutant 1-PVM (S2P M71V K145M) to increase activity on L-tryptophan, tryptoline, and desbromodeformylflustrabromine. Variants were also tested for their ability to chlorinate tryptoline at different catalyst loads (FIGS. 5A-5E).

(41) TABLE-US-00004 TABLE 4 Ability of WT RebH and Variants to Chlorinate Native and Non-Native Substrates (% conversion) 2-methyl L-tryptophan tryptamine tryptoline (0.2% load) (1% load) (4.7% load) wt 27 39 15 S2P 40 63 62 S2P M71V K145M 42 59 50 S2P M71V K145M N467T 69 63 90 S2P M71V K145M F458S 39 24 9 S2P M71V K145M T394M 57 60 51 S2P M71V K145M 29 9 6 E423D E461G S2P M71V K145M 27 41 41 T348A L453P A476T S2P M71V K145M D264G 46 62 30 S2P F465C 15 63 30

(42) TABLE-US-00005 TABLE 5 Ability of WT RebH and Variants to Halogenate Native and Non-Native Substrates (% conversion) L-tryptophan tryptoline (0.2% load) (4.7% load) wt 39 8 S2P M71V K145M 57 22 S2P M71V K145M N467T 99 44 S2P M71V K145M N467T L380F 79 40 S2P M71V K145M N467T D101G K237E 60 37 S2P M71V K145M N467T N470S 41 98 S2P M71V K145M N467T F171I T283A 94 33 S2P M71V K145M N467T L114P 31 42 S2P M71V K145M N467T G112S 50 76 S2P M71V K145M N467T V256I 83 45

(43) TABLE-US-00006 TABLE 6 Ability of WT RebH and Variants to Halogenate Native and Non-Native Substrates (% conversion) Debromo- L- Tryptoline desformyl- tryptophan (0.5% flustrabromine Evodiamine (0.2% load) load) (5% load) (5% load) wt 53 3 0 2 S2P M71V K145M (1-PVM) 43 6 0 9 S2P M71V K145M N467T (2-T) 53 8 0 8 S2P M71V K145M N467T G112S 22 64 6 26 N470S (3-SS) S2P M71V K145M N467T N470S 48 50 29 27 S2P M71V K145M N467T N470S 42 43 48 28 A442V (4-V) S2P M71V K145M N467T N470S 38 D203G

(44) Table 6 presents data obtained from a second set of the activity experiments described in Table 5, in which the activities of the mutants were tested against L-tryptophan, Tryptoline, Debromo-desformyl-flustrabromine, and Evodiamine.

(45) Some of the mutants described above were able to chlorinate a broad range of substrates. These substrates are illustrated in FIG. 8.

(46) Potentially key mutations: FIG. 8 indicates that mutant 4-V accepts a wide range of very large substrates, including carvedilol and yohimbine. Mutation A442V appears to be broadly useful for large substrates, especially those with MW>>200 g/mol (the approximate MW of L-tryptophan). The top four substrates shown in FIG. 8, all of which have tricyclic tryptoline-like structures, were chlorinated best by mutant 3-SS. For these substrates, the G112S and N470S mutations appeared to be especially important. The high activities obtained with mutant 4-V applied to large substrates worked well with the removal of the G112S mutation, suggesting that this mutation can be detrimental to high activity on large substrates.

(47) The activities of mutants 4-V and 3-SS were compared to the activity of the wild-type RebH for each substrate shown in FIG. 8. These ratios are listed in Table 7:

(48) TABLE-US-00007 TABLE 7 Substrate Mutant Mol % Enzyme Activity Ratio.sup.[a] Tryptoline (2) 3-SS 0.5 65.5 Eleagnine (5) 3-SS 0.5 67.1 Pinoline (6) 3-SS 0.5 2.0 Tetrahydroharmine (7) 3-SS 5 17.6 Debromo-dFBr (3) 4-V 5 N/A.sup.[b] Yohimbine (8) 4-V 5 N/A.sup.[b] Evodiamine (4) 4-V 5 16.5 Pindolol (9) 4-V 0.2 1.3 Carazolol (10) 4-V 0.2 4.9 Carvedilol (11) 4-V 0.5 8.2 Table 1 .sup.[a]Activity ratio is ratio of conversion seen with mutant tested vs. WT. Reaction conditions were those shown in Scheme 2. .sup.[b]WT showed no detectable activity, and thus a ratio cannot be determined.

(49) Solvent toleranceSolvent tolerance analyses were run under the same conditions as the characterization of evolved RebH mutants, with the exception that 30% DMSO was added as a co-solvent (Table 8).

(50) TABLE-US-00008 TABLE 8 Solvent Tolerance, 30% DMSO as co-solvent, 1% load (% conversion) wt 27 N75K 17 E96V 19 F312L 16 S2P 28 S2P, M71V 67 S2P, T213A 29 S2P, K145M 33 S2P, D203A 48 S2P, F396Y 44 F396Y 7 M71V 33 M71T 23 M71A 65 M71C 35 M71V, T213A 20

(51) Crystal structure of thermostable 3-LSRThe crystal structure of 3-LSR (PDB ID 4LU6) was solved by molecular replacement using wild-type RebH (PDB ID 2OAM) as the search model. The model was refined to 3.05 with a final R.sub.work=18% and R.sub.free=24%. Wild-type RebH and 3-LSR are similar overall with a backbone root mean square deviation (rmsd) of 0.32 . The differences in the structures are localized in the eight amino acid changes between the two enzymes.

(52) Investigating the location and nature of the mutations in the structure of 3-LSR may provide a molecular basis for the increase in thermostability and T.sub.opt. Mutation Q494R is located on the protein surface and converts the neutral side chain of glutamine into the positively charged side chain of arginine. Increasing the amount of surface charge is a deterrent to protein aggregation. The serine-to-proline mutation of S2P is located right at the N-terminus, and proline residues generally increase protein rigidity by decreasing the flexibility of the polypeptide chain. Indeed, the five other RebH structures in the PDB start their models at amino acid number two or three; in 3-LSR, the electron density map extends to amino acid number one, indicating increased order at the N-terminus. The increased rigidity of the N-terminus might also help stabilize the protein by preventing it from acting as a fraying point for thermal denaturation. Mutation K145M is located near the surface of the protein and in the area of two arginine residues (FIG. 4). Wild-type RebH increases the density of positive charge in the area with lysine, and 3-LSR might be stabilized by reducing this density by substituting a methionine at this position. Also, the side chain of methionine adopts a conformation that increases its packing with neighboring residues, which might enhance thermostability.

(53) Changing the regioselectivity of RebH through directed evolutionThrough methods analogs to the directed evolution of RebH for increased thermostability, the regioselectivity of RebH has been altered on the unnatural substrate tryptamine (ratio of 7-6-5 selectivity, Table 10). This is important in broadening the scope of RebH to chlorination of CH bonds within a molecule beyond its natural regioselectivity, and can be applied to changing the regioselectivity on other substrates in the future. The first variant for this evolution was RebH-N470S.

(54) TABLE-US-00009 TABLE 9 Variant with Increased Activity on Tryptamine N470S Variant with Altered Selectivity on Tryptamine - Rd 1 N470S, S448P Variants with Increased Activity on Overlap Extension PCR-Recombined Variant From Tryptamine - Rd 2 Second Round Variants N470S, S448P, L380F, Q494R N470S, S448P, L380F, Q494R, R509Q N470S, S448P, R509Q Variant with Altered Selectivity on Tryptamine - Rational point mutation N470S, S448P, L380F, Q494R, R509Q, Y455W Variant with Increased Activity on Tryptamine - Rd 3 N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P Variant with Altered Selectivity on Overlap Extension PCR-Recombined Variant From Tryptamine - Rd 3 Third Round Variants N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L F111L Variants with Increased Actvity on Tryptamine - thermostability mutation Overlap Extension PCR-Recombined Variants From additions Thermostability Mutants N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, S110P, F111L, S130L F111L, S130L, N166S N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L, N166S Variants with Altered Selectivity on Overlap Extension PCR-Recombined Variants From Tryptamine - Rd 4 Fourth Round Variants N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, S110P, F111L, S130L, N166S, T322I, F458L, F111L, S130L, N166S, F465L F465L, V481A N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, S110P, F111L, S130L, N166S, I52T, T496A F111L.fwdarw.L111S, S130L, N166S N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, S110P, F111L.fwdarw.L111S, S130L, N166S, A58V F111L.fwdarw.L111S, S130L, N166S, F465L N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L, S130L, N166S, F465L, I52T N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L.fwdarw.L111S, S130L, N166S, I52T N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L.fwdarw.L111S, S130L, N166S, F465L, I52T Variants with Altered Selectivity on Tryptamine - NDT library N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L.fwdarw.L111S, S130L, N166S, G112D, L113N N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L.fwdarw.L111S, S130L, N166S, G112D, L113D Variants with Altered Selectivity on Overlap Extension PCR-Recombined Variant From Trytpamine - Rd 5 Fifth Round Variants N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P.fwdarw.P110L, F111L, S130L, N166S, F465L, S110P.fwdarw.P110L, F111L, S130L, N166S, F465L, I52T, I52T, K145R, A476V N470S, S448P, L380F, Q494R, R509Q, Y455W, N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L, S130L, N166S, F465L, I52T, S110P.fwdarw.P110L, F111L, S130L, N166S, F465L, I52T, K145R, A476V K187R, F396L N470S, S448P, L380F, Q494R, R509Q, Y455W, S110P, F111L, S130L, N166S, F465L, I52T, K187R, F396L S110P.fwdarw.P110L and F111L.fwdarw.L111S denote secondary mutations, which means the S110P and/or F111L mutations from one round were further mutated to give the respective P110L and L111S mutations. The effective mutations from WT are S110L and F111S.

(55) TABLE-US-00010 TABLE 10 Ratio of % Loading of RebH 7-6-5 Best Actvity and Selectivity Variants relative to tryptamine % Conversion selectivity N470S 1.67 86 99-1-0 N470S, S448P 1.67 55 94-4-2 N470S, S448P, L380F, Q494R, R509Q 1.67 91 94-4-2 N470S, S448P, L380F, Q494R, R509Q, Y455W 1.67 76 91-6-3 N470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 50 85-10-5 S110P, F111L N470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 68 85-10-5 S110P, F111L, S130L, N166S N470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 24 25-75-0 S110P, F111L.fwdarw.L111S, S130L, N166S N470S, S448P, L380F, Q494R, R509Q, Y455W, 5 33 34-35-31 S110P, F111L, S130L, N166S, F465L, I52T N470S, S448P, L380F, Q494R, R509Q, Y455W, 1.67 51 18-82-0 S110P, F111L.fwdarw.L111S, S130L, N166S, G112D, L113N N470S, S448P, L380F, Q494R, R509Q, Y455W, 5 29 25-36-39 S110P.fwdarw.P110L, F111L, S130L, N166S, F465L, I52T S110P.fwdarw.P110L and F111L.fwdarw.L111S denote secondary mutations. The S110P and/or F111L mutations from one round were further mutated to give the respective P110L and/or L111S mutations. The effective mutations from WT RebH are S110L and F111S.

REFERENCES

(56) Adams, P. D., Afonine, P. V., Bunkczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffher, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) Acta Crystallographica, D66, 213-221. Anderson, J. L. R., Chapman, S. K. (2006) Molecular Biosystems, 2, 350-357. Blasiak, L. C. and Drennan, C. L., (2009) Accounts of Chemical Research, 42, 147-155. Butler, A. and Sandy, M. (2009) Nature, 460, 848-854. Emsley, P. and Cowtan, K. (2004) Acta Crystallographica, D60, 2126-2132. Heckman, K. L. and Pease, L. R. (2007) Nature Protocols, 2, 924-932. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni L. C., and Read. R. J. J. (2007) Applied Crystallography, 40, 658-674. Otwinowski, Z. and Minor, W. (1997) Methods in Enzymology, 276, 307-326. Payne, J. T., Andorfer, M. C. and Lewis, J. C. (2013) Angewandte Chemie International Edition, 125, 5379-5382. Petsko, G. A. (2001) Methods in Enzymology, 334, 469-478 Vaillancourt, F. H., Yeh, E., Vosburg, D. A., Garneau-Tsodikova, S. and Walsh, C. T. (2006) Chemical Reviews, 106, 3364-3378.