Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study

Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein dynamics and how the introduced cofactors alter the protein scaffold structure. Here we present the structures and catalytic activity of novel copper ArMs capable of (R)- or (S)-stereoselective control, utilizing a steroid carrier protein (SCP) scaffold. To incorporate 2,2′-bipyridine (Bpy) into SCP, two distinct strategies were employed: either Bpy was introduced as an unnatural amino acid (2,2′-bipyridin-5-yl)alanine (BpyAla) using amber stop codon expression or via bioconjugation of bromomethyl-Bpy to cysteine residues. The resulting ArMs proved to be effective at catalysing an enantioselective Friedel–Crafts reaction with SCP_Q111BpyAla achieving the best selectivity with an enantioselectivity of 72% ee (S). Interestingly, despite using the same protein scaffold, different attachment strategies for Bpy at the same residue (Q111) led to a switch in the enantiopreference of the ArM. X-ray crystal structures of SCP_Q111CBpy and SCP_Q111BpyAla ArMs with bound Cu(ii) ions unveiled crucial differences in the orientation of the catalytic centre. Combining structural information, alanine scanning studies, and computational analysis shed light on the distinct active sites of the ArMs, clarifying that these active sites stabilise the nucleophilic substrate on different sides of the electrophile leading to the observed switch in enantioselectivity. This work underscores the importance of integrating structural studies with catalytic screening to unravel the intricacies of ArM behaviour and facilitate their development for targeted applications in biocatalysis.


General Remarks
Commercially available chemicals used in the study were purchased from commercial suppliers (Sigma-Aldrich, Fluorochem).
NMR spectra were recorded on a Bruker Avance III 500 or 400 MHz spectrometer at 300 K.Chemical shifts are reported in parts per million (ppm) and referenced to the residual solvent peaks: CDCl3 ( 1 H: δ 7.26 ppm), D2O (1H: δ 4.79 ppm).Coupling constants (J) are reported in Hertz (Hz) and were calculated using MestReNova (version 14.2.0) and rounded to the nearest 0.1 Hz.The following abbreviations (and their combinations) are used to label the multiplicities: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet) as observed in the spectra.The molecular weight of the protein mutants was determined using electrospray ionization mass spectroscopy (ESI-MS) analysis on a Waters Synapt G2 with Waters Acquity I-Class UPLC (LC-MS).ICP-MS data was collected on a Agilent 7500 ce.Enantiomeric ratio of the reaction were analysed using HPLC (Chiralpak AD-H, 250 x 4.6 mm) at a flow rate of 1 ml/min using n-hexane:iPrOH 85:15 as a solvent and the products were detected at wavelength of 280 nm.Chemically competent E. coli DH5α cells were used for cloning, E. coli BL21 and Rosetta 2 DE3 cells for protein expression.Primers for site-directed mutagenesis were purchased from Sigma Aldrich, USA and IDT, USA.PCR Master Mix and DpnI were purchased from NEB, USA.GeneJet Plasmid Minikit for plasmid extraction was purchased from ThermoFisher Scientific, USA.HisTrap HP Ni-charged immobilized metal affinity column (IMAC) was purchased from GE Healthcare, USA.The mass of proteins was calculated using the Expasy ProtParam tool (http://web.expasy.org/protparam/).

Molecular Biology
All the described research has been performed using SCP_2L as a protein scaffold.The metal binding moiety was introduced to the protein scaffold by either bioconjugation of cysteine mutants with the Cys codon at A100C, V83C, Q111C; or by genetic code expansion using amber stop codon suppression: TAG mutants with A100TAG, V83TAG, and Q111TAG.The sequences for TAG mutants were codonoptimized for expression in E. coli.

SCP_2L Cysteine mutants[1]
The Cys mutants have an N-terminal His-tag.The portion removed by TEV protease is highlighted in red, and the TEV protease recognition site highlighted in italics.

Gene optimization and SCP_2L TAG mutants
The SCP_A100C gene was synthesized at Genescript and included a C-terminal TEV site and His6tag.The codons were optimized to E. coli codon bias.The gene was cloned into the vector pET28.

Site-directed mutagenesis
The 'wild-type' SCP containing no cysteine and a C-terminal His6 tag, C-ter His SCP_C100A, was prepared by site directed mutagenesis.The mutant A100TAG was prepared using site-directed mutagenesis directly from the E.coli optimised C-ter His SCP_A100C gene, whilst the V83TAG, and Q111TAG mutants of SCP_2L were prepared from C-ter His SCP_C100A.The primers used for mutagenesis are summarized in Table S1.E. coli DH5α were transformed and plated, a single colony of freshly transformed cells was cultured overnight in 5 ml LB media containing 50 μg/ml of kanamycin at 37 °C, 200 rpm.The plasmid DNA was isolated using plasmid extraction kit and sent for sequencing with 20 μL of the plasmid (concentration 30 ng/μL) to Dundee DNA Sequencing and Services, using facility provided T7 primer.

Protein expression and purification
Cysteine mutants of the SCP-2L scaffold were expressed following the protocol in [1], repeated here for clarity.For the expression of A100C, V83C, Q111C, chemically competent E. coli Rosetta DE3 cells were transformed with pEHISTEV::dΔhΔSCP-2L plasmid and plated overnight.A single colony of freshly transformed cells was cultured overnight in 50 ml LB media containing 50 μg/ml of kanamycin and 34 μg/ml of chloramphenicol at 37 °C, 200 rpm.Starter cultures (20 ml) were used for inoculation of 1000 ml of PB media (Production Broth medium; containing 20 g/L tryptone, 10 g/L yeast extract, 5 g/L dextrose, 5 g/L NaCl, 8.7 g/L K2HPO4, pH 7.0) supplemented with 50 μg/ml of kanamycin and 34 μg/ml of chloramphenicol and grown at 37 °C, 200 rpm to an OD600nm ~ 0.6 (~ 2.5 h).Protein expression was induced with the addition of IPTG to a final concentration of 0.2 mM.The induced cultures were incubated for 18 h at 16 °C, 200 rpm.
For the expression of TAG mutants, chemically competent E.coli BL21 cells containing pEVOL-PylRSAlaBpy/tRNA CUA were transformed with pET28_optimised_ΔSCP-2L plasmid plated overnight.
A single colony of freshly transformed cells was cultured overnight in 50 ml LB media containing 50 μg/ml of kanamycin and 34 μg/ml of chloramphenicol at 37 °C, 200 rpm.Starter cultures (20 ml) were used for inoculation of 1000 ml media supplemented with 50 μg/ml of kanamycin and 34 μg/ml of chloramphenicol and grown at 37 °C, 200 rpm to an OD600nm ~ 0.6 (~ 2 h).Prior to induction of protein expression, racemic BpyAla was added with a final concentration of 0.5 mM and the temperature lowered to 30 °C.Protein expression was induced with the addition of IPTG with a final concentration of 0.5 mM and L-arabinose with a final concentration of 0.2% w/v.The induced cultures were incubated overnight 14 h at 30 °C, 200 rpm.
The cells of both induced cultures were subsequently harvested by centrifugation (4200 rpm for 15 min at 4 °C).Pelleted cells were resuspended in 20 ml PBS and pelleted by centrifugation (4200 rpm for 15 min at 4 °C), the buffer was discarded, and the cell pellets were frozen at -20 °C.
The purification protocol has been described in [1] and repeated here for clarity.After defrosting the cell pellets, 20 ml of lysis buffer (50 mM Tris-HCl, 20 mM imidazole, 150 mM NaCl, 0.5 mM benzamidine, pH 8) was added to resuspend the cells from 1 L of culture.Then 20 mg of lysozyme, 1 mg of DNase I, and 1 ml of 1 M MgCl2 were added to the lysate and incubated for 1 h.The lysate was subjected to sonication (1 min, 90 % power with 5 s pulses).Cell lysates were cleared by centrifugation (4200 rpm for 1 h at 4 °C) and supernatant, filtered with 0.45 μm filter (Millipore, Merck), was subjected to IMAC 5 mL HisTrap HP column at a constant flow-rate of 5 ml/min equilibrated with 5 cv of wash buffer (30 mM Tris-HCl, 150 mM NaCl, pH 8 containing 20 mM imidazole).The column was first washed with 5-10 cv of wash buffer.The His-tagged variants were eluted with 6 cv of 50% elution buffer (wash buffer containing 330 mM imidazole).Eluted proteins were dialyzed against 5 L of buffer solution (wash buffer with 10 mM imidazole) at 4 °C overnight.To remove the His-tag, 0.014 equivalents of TEV-protease and final concentrations of 1 mM DTT and 0.5 mM EDTA were added and incubated for 6 h at room temperature.After TEV cleavage, the protein solution was subjected to IMAC 5 ml HisTrap HP column equilibrated with wash buffer at a constant flow of 5 ml/min.The flowthrough that contained SCP-2L variants with His-tag cleaved was collected and analysed by SDS PAGE (RunBlue 4-12% Bis-Tris Gel, Expedeon; NuPAGE MES SDS Running Buffer, Life Technologies; Invitrogen Mark12 TM unstained standard).The protein concentrations were determined using A280 and the extinction coefficients corrected for each mutant for the presence of BpyAla (ε=14000) (Table S2), the concentration of modified Cys mutants and TAG mutants was determined using the Mw and extinction coefficient.

Bioconjugation of Cys Mutants
Purified protein samples of Cys mutants were buffer-exchanged into HEPES (0.1 M, 50 mM NaCl, pH 8.5) and diluted to the concentration of 100 μM.To the protein solution, 10 equivalents of the Br-Bpy complex were added and the bioconjugation reaction took place for 1 h at 25 °C.The reaction was stopped with buffer exchange into MES buffer (20 mM, 50 mM NaCl, pH 6) using Amicon 10kDa Ultra Centrifugation Filters (Merck, USA).The bioconjugation of Cys mutants was confirmed using ESI-MS analysis.The protein identity further analysed with SDS PAGE.

ICP analysis
ICP analysis was used to accurately determine the copper concentrations in samples to determine the stoichiometry of Cu binding to the Bpy moiety in the protein.SCP_Bpy conjugates in MES buffer (20 µM, determined using A280 and the extinction coefficients given in Table S3) were incubated with one equivalent of metal salt in order to form the 1:1 complex SCP_2L:Cu 2+ and then buffer exchanged threetimes to remove any residual copper using Amicon 10kDa Ultra Centrifucation filters (Merck), never exceeding the initial volume.Duplicate samples for ICP analysis were prepared as following: to 100 µL of the resulting metalloproteins soultion in a 15 ml falcon tube was added nitric acid (100 µL, 69% Aristar for trace analysis) and heated at 70 °C for 5-6 h.It was diluted to 3 ml to give concentration of less than 5% of nitric acid.
Trace metal analysis was performed by the microanalysis service at the University of Edinburgh on an Agilent 7500ce (with octopole reaction system), employing an RF forward power of 1540 W and reflected power of 1 W, with argon gas flows of 0.81 L min -1 and 0.20 L min -1 for carrier and makeup flows, respectively.Sample solutions were taken up into the Micro mist nebuliser by peristaltic pump at a rate of approximately 1.2 mL min-1.Skimmer and sample cones were made of nickel.The instrument was operated in spectrum multi-tune acquisition mode and three replicate runs per sample were employed.
Yields of the catalytic reactions are based on peak areas 280 nm using 2-phenylquinoline as internal standard.The following mutants were expressed and purified: Q111Bpy (for the control), Q111BpyAla_F34A, Q111BpyAla_L114A, Q111BpyAla_M112A, Q111BpyAla_Q108A, Q111BpyAla_D88A, Q111BpyAla_Q90A, Q111BpyAla_V82A, Q111BpyAla_K115A as described in 3 Protein expression and purification.

Size exclusion chromatography of SCP_Q111BpyAla
Prior to the crystallization screen the proteins underwent a polishing purification step using gel filtration chromatography.The protein sample was concentrated to a volume of ~ 1 ml.The concentrated sample was loaded onto a HiPrep Superdex S 75 HR column (Vt ~ 120 ml, 2.6 x 60 cm) (GE Healthcare, Uppsala, Sweden) pre-equilibrated with size-exclusion buffer (20 mM PIPES, 1 mM EDTA, 1 mM sodium azide, 150 mM NaCl, pH 7.5).

Crystallization
Prior to crystallisation, proteins were concentrated by centrifugal filtration in buffer containing 20 mM PIPES, 1 mM EDTA, 1 mM sodium azide, 150 mM NaCl, pH 7.5, 0.15 mM Triton X-100 to 5 mg/ml (SCP_Q111CBpy; 120 amino acids) and 5 mg/ml (SCP_Q111BpyAla; 128 amino acids).Crystals of SCP_Q111Cbpy were grown by hanging-drop vapour diffusion at 4 °C using the same precipitant used to grow crystals of the wt SCP_2L [16]: 2.3M ammonium sulphate, 100 mM citric acid pH 5.9 and 200 mM NaCl.Drops contained 1 µL of SCP_Q111CBpy protein and 1 µL of precipitant solution.Crystals with approximate dimensions 0.05 mm x 0.05 x 0.05 mm grew within 14 days.SCP_Q111BpyAla did not spontaneously crystallise in these or similar conditions, so a few SCP_Q111CBpy crystals were   11 Synthesis of BpyAla

1-(2-pyrdylacetyl)pyridinium iodide, S1
Anhydrous pyridine (400 mL) was added to iodine (75.6 g, 300 mmol) under Ar atmosphere.Acetyl pyridine (33.6 mL, 300 mmol) was added, the solution was heated to 125 o C for 4 h.The heating was then turned off and the solution was stirred at r.

5-bromomethyl-2,2-bipyridine, S3
Cyclohexane (400 mL) was added to 5-methyl-2,2-bipyridine S2 (15 g, 88.2 mmol) and degassed with Ar.NBS (29.9 g, 168 mmol, 1.75 eq, recrystalised from MeCN) was added.The solution heated to 80 °C with vigorous stirring (1000 rpm).AIBN (790 mg, 4.8 mmol, 0.05 eq) was added portionwise over about 20 minutes and heated for 3 h further causing a black precipitate to form.The solution was allowed to cool slightly and the solution was decanted away from the black precipitate, the precipitate was rinsed with 2x30 mL hot cyclohexane and then concentrated on the rotovap to give a yellow/brown oil.The flask was put in a ice bath whit stirring and cold hexane (50 ml) was added in one portion .The pale yellow precipitate was collected by vacuum filtration, washed with cold hexane (2 x 20 mL) and dried over vacuum giving the product S3 as a pale yellow solid (12.9 g, 59 % yield).Analytical data in accordance with literature [6].

Diethyl 2-(2,2´-bipyridin-5-ylmethyl)-2-acetamidomalonate, S4
To a 2-neck 250 mL flask containing NaH (2.2 g, 55 mmol, 60% in mineral oil) under argon at 0 o C was added anhydrous DMF (10 mL).Diethylacetamidomalonate (10.8 g, 50 mmol, 1 eq.) was dissolved in anhydrous DMF (20 mL) and added to the NaH solution dropwise with stirring.As the addition neared completion, the solution became viscous and stirring slowed.The cooling bath was removed causing the solution to become fluid once more.Addition of the malonate was continued ensuring the temperature of the reaction did not go above 20 o C.After addition was complete the solution was stirred at r.t. for 30 min.
The solution was cooled back to 0 o C on ice.Bromomethy-2,2-bipyridine S3 (12.35 g, 50 mmol) was dissolved in DMF (10 mL) and added dropwise to the malonate solution.After addition the reaction was stirred at r.t. for 5 h.The solution was cooled back to 0 o C on ice and water (150 mL) was added with vigorous stirring.The precipitate that formed was collected by vacuum filtration.The solid was taken up into CHCl3 (50 mL) and stirred with activated charcoal (approx.2.5 g).The solution was filtered through celite and concentrated under vacuum oven giving S4 as a white solid (11.6 g, 60% yield).Analytical data in accordance with literature [5].
Due to the change in site of the His6 tag and TEV protease recognition site from the N to the C termini of the protein the sequence varies slightly between the cysteine and TAG genes Optimized DNA sequence of C-ter His SCP_A100C

Figure S1 :
Figure S1: SDS PAGE purity analysis of SCP_2L purifications.SCP_V83C: 1 Ladder, 2 Flowthrough of the first Ni-column purification, 3 After first Ni-column purification and dialysis, 4 Flowthrough of the second Ni-column purification, 5 Pure protein after second Ni-column purification, SCP_Q111C: 6 Ladder, 7 Flow-through of the first Ni-column purification, 8 After first

Figure S2 :
Figure S2: Deconvoluted and raw spectra, high-resolution mass spectroscopy for all protein mutants after purification used in this study.Comparison of calculated (Mcalc) and observed (Mobs) masses confirms the identity of each variant and successful incorporation of Cys and BpyAla.For the analysis of data we used MassLynx software.

Figure S4 :6
Figure S4: Deconvoluted, high-resolution mass spectroscopy for all protein mutants after modification with Br-Bpy used in this study.Comparison of calculated (Mcalc) and observed (Mobs) masses confirms the identity of each variant and successful Cys modification.

Figure S15 :
Figure S15: Close-up view of Cu(II) bound to SCP_Q111BpyAla, showing octahedral coordination of the four water molecules (red spheres) and the N1 and N2 of BpyAla111.The grey mesh represents the 2Fo-Fc electron density map (contoured at 1).

Table S2 :
Extinction coefficients of the mutants.

Table S3 :
ICP analysis of Cu binding to SCP_Q111BpyAla

Table S4 :
pH effect on enantioselectivity and yield of F-C reaction.
The mutations were prepared using site-directed mutagenesis as described (2.3 Site directed mutagenesis).

Table S5 :
Primers used to make SCP_Q111BpyAla Alanine mutants.