A 2‐Tyr‐1‐carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase

Abstract N,N‐dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2β2 or (α2β2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side‐chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.


Protein expression and purification
parDMFase was purified from both the wild-type and recombination sources. Cell pellets of Paracoccus sp. strain DMF were washed twice with buffer A (Tris-Cl 50 mM, NaCl 50 mM, dithiothreitol (DTT) 1 mM, pH 7.2 at 4 ºC) and re-suspended in cell lysis buffer B (buffer A, NaCl 200 mM, MgCl2 10 mM, lysozyme 0.3 mg/mL, DNase 0.01 mg/mL, glycerol 10% v/v) and incubated at 4 °C for a minimum of 4h before lysis by sonication. The cell extract was obtained by centrifugation of the lysate at 20000*g for 45 min at 4 ºC. Purification was achieved by high-salt precipitation followed by ion-exchange chromatography. Ammonium sulfate salt was added to the cell extract culture (50-70% saturation range). The resulting precipitate was collected by centrifugation at 10,000*g for 10 min and re-dissolved in buffer A and dialyzed overnight against three exchanges of the same buffer. The dialyzed protein solution was incubated at 45 °C for 15 min on a heating block and was immediately transferred into ice, and heat-denatured aggregated protein fractions were removed by centrifugation at 12000*g for 15 min at 4 °C. Two-step anionexchange chromatography was performed for further purification. The protein solution was injected into a HiPrep Q-FF (16/10) column (GE Healthcare Life Sciences) pre-equilibrated with buffer A.
Column was washed with 10 CV buffer containing 0.2 M NaCl and protein was eluted with 20 CV of the same buffer with a linear gradient of 0.2 to 0.6 M NaCl. A flow rate of 1 mL/min was maintained throughout the purification process. The purified fractions as determined by SDS-PAGE (12%) were pooled and concentrated using a Millipore Amicon ultra 100K device. Dialysis was performed for 6h against buffer A and re-injected into the same column. Protein was eluted on a linear gradient of NaCl (0.3 to 0.6 M). This step was found to be essential for purification of the native parDMFase to homogeneity. Peak fractions from the anion-exchange column were pooled, concentrated, and was followed by size exclusion chromatography (SEC) with a Superdex column (S-200, 16/60, GE Life Sciences) pre-equilibrated with buffer C (Tris-Cl 50 mM, NaCl 250 mM, DTT 1 mM, pH 7.2 at 4 ºC). The parDMFase was eluted with the same buffer at the flow rate of 1 mL/min. All the peak and pooled fractions were tested for enzymatic activity.
The parDMFase constructs fused to a C-terminal His6 tag were expressed in E. coli BL21 star (DE3) cells. Cells were grown in LB medium supplemented with ampicillin (100 μg/ml) at 37 °C until they reached an OD600nm of ~0.6. Protein overexpression was induced by the addition of 0.5 mM IPTG and cultures were grown for 6h at 37°C. Cell pellets were pooled by centrifugation at 4000*g for 15 min, and resuspended in lysis buffer B and sonicated. The lysate was cleared by centrifugation at 14000*g for 45 min at 4°C. Further purification of the recombinant enzyme was performed using steps as described for native enzyme purification. SEC elution fractions corresponding to parDMFase were either used immediately or flash frozen in liquid nitrogen with 20% glycerol and stored at -80 °C until further use.

Protein concentration determination
Protein concentration at each purification step was estimated by the Bradford method using bovine serum albumin (BSA) as a standard [1] .

Steady-state enzyme assay
Amidohydrolase activity of parDMFase towards N-substituted amides was determined using an alkylamine-specific colorimetric assay (Cullis and Waddington 1956). The standard enzyme assay with DMF contained 45 µL of 50 mM buffer C, 50 µL enzyme solution, and 5 µL of 3M DMF.
The reaction was carried out in tightly closed microcentrifuge tubes for 30 min at 37°C. The reaction was quenched by the addition of 25 µL trichloroacetic acid (TCA) solution (15%, w/v).
After centrifugation at 10000*g for 20 min, 100 µL of the reaction mixture was added to 1 mL of carbonate buffer (sodium tetraborate hexahydrate (9.53 g/L) and sodium carbonate (5.3 g/L) solution (pH 9.8)), 0.25 mL freshly prepared sodium nitroprusside (Na2[Fe(CN)5NO]) solution (1% w/v), and 0.25 mL acetaldehyde solution (l0% v/v). The dimethylamine (DMA) formed in the enzymatic reaction leads to a change in the absorbance, which was measured after 20 min at 580 nm. Spectrophotometric measurements were carried out with a blank solution in which DMF was added to the enzyme mixture after the enzyme inactivation step with TCA. The concentration of DMA produced upon enzymatic reaction was determined from a standard curve calibrated with known concentrations of DMA.

Isothermal Titration Calorimetric (ITC) enzyme assay
To perform the real-time ITC enzyme kinetic assay, two sets of experiments were carried out. In single injection mode, the total molar enthalpy (ΔHº) was extrapolated by titrating 25 µL from a single injection of a relatively low concentration of substrate (25 mM) to a relatively high concentration (25 nM, 203.7 µL) of parDMFase in the cell (Microcal ITC200, GE-Healthcare). In the multiple injection mode, enzymatic reaction was initiated by a series of injections of a relatively high concentration of DMF (100 mM, 2 µL, 20 injections) with a relatively low concentration of enzyme (25 nM) in the cell. All experiments were performed in buffer C at 37 °C in high-feedback mode with a stirring speed of 1000 rotations per minute and a filter time of 4s. A pre-injection delay of 600 s was applied in order to establish a steady baseline. A plot of heat rate vs. substrate concentration points was obtained in order to calculate the kinetic parameters after a baseline correction. The ΔHº value was determined in terms of the shift in thermal power that occurred from the conversion of the substrate before returning to its pre-equilibrated base-line [2] . The Michaelis-Menten enzyme parameters KM, kcat, and steady-state heat (pseudo first order) rate were derived from the enzyme that was titrated with increasing concentrations of substrate. The kinetic experiments were carried out in duplicate. A non-linear regression fitting the Michaelis-Menten equation (GraphPad Prism v6.0 software) was used to calculate the kinetic parameters (Km and kcat).

H-NMR spectroscopy of enzyme kinetics for substrate specificity
Hydrolase activity of parDMFase towards DMF substrate analogs including formamide, acetamide,

Thermal shift assay
Protein unfolding and stability were determined as a function of temperature. In experimental studies, a label-free thermal shift assay was performed using Tycho NT. 6 (NanoTemper Technologies). Pre-dialyzed protein and mutant samples were diluted (~0.5 mg/mL) in appropriate buffer conditions and run in duplicates in capillary tubes. Intrinsic fluorescence from tryptophan and tyrosine residues was recorded at 330 nm and 350 nm while heating the sample from 35°C to 95°C at a ramp rate of 30°C/min. The ratio of fluorescence (350/330 nm) and the inflection temperature (Ti) were calculated using Tycho NT. 6 software, which provides Tm (melting point), the temperature at which 50% of the measured protein is unfolded.

Electron microscopy of parDMFase
Initial images of parDMFase data at 2-3 mg/ml in 50 mM NaCl were collected using Quantifoil holey carbon grids (R 0.6/1, Au 300 mesh) with blotting and freezing accomplished with a manual plunger in a cold room. This initial data was collected with Titan Krios at MRC LMB, Cambridge, and Falcon 2 detector in integration mode with the EPU software. Images showed that there were two populations, and both these were picked and subjected to reference-free 2D classification.
Initial models of both the populations were individually generated either with EMAN2 [3] or by Stochastic gradient descent within RELION [4] with C2 symmetry imposed. The refinement of the dimer population obtained in the integration mode gave a reconstruction of ~7 Å, indicating that the protein behaved well and a high resolution structure can be obtained. All the data described here were collected from the enzyme obtained by recombinant expression. Enzyme purified from native source showed similar maps (data not shown).
Subsequently, data were collected with a Falcon 3 detector in counting mode at 1.07 Å sampling and images were exposed for 60 seconds with a total accumulated dose of ~27 e/Å 2 and dose fractionated into 75 frames, with each frame having a dose ~0.3 e. The images were grouped into 25 frames, resulting in ~1 e/frame, and Unblur [5] was used for alignment. This initial processing was performed in Relion 2.0 [6] . The summed images were then used for automated particle picking with Gautomatch with template derived from previous data collection, and CTF was estimated with Gctf [7] . Particles were extracted with a box size of 320 pixels and subjected to 2D classification, 3D auto-refinement, per particle motion-correction, B-factor weighting, and refinement. Further 3D classification was used to improve the quality of the maps by removing bad particles. This resulted in a 3.4 Å map for the dimer and 3.8 Å for tetramer. We noticed that the views in the tetramer were not diverse, may be because of thinner ice.
During this period, we observed that the oligomeric state of the enzyme is affected by salt and subsequently we imaged parDMFase in three different salt concentrations (no salt, 200 and 500 mM NaCl). All of the data presented here were collected at the National CryoEM facility in Bangalore with a Falcon 3 detector in counting mode at 1.07 Å sampling and 60s exposure. The grids for these datasets were Au 300 mesh either R0.6/1 or 1.2/1.3 and made with Vitrobot Mark IV at 100% relative humidity and 16C. The grids were blotted for 3.5 seconds. The datasets were processed with Relion 3.0, including the whole frame alignment and dose-weighting with Relion's own algorithm. Particle picking and CTF estimation were performed using Gautomatch and Gctf.
Particles were extracted with a box size of 320 pixels and subjected to 2D classification, 3D auto-refinement, per particle CTF refinement, B-factor weighting with Bayesian polishing and refinement, and subsequent 3D classification [8] . The data sets of mutants Y440A and E521A were processed similarly. The local resolution of the maps was estimated using Resmap [9] . Model building was performed with Coot [10] , and the model was refined with phenix.real_space_refine [11] .
Although there are non-protein densities in the map, we modelled only one water molecule at the active site and the rest were not modelled. Details of the EM data and model quality are presented in Supporting information Table 2.

Crystallization, refinement and model building
The purified native and recombinant parDMFase were concentrated to 10 and 5 mg ml -1 . Initial crystallization trials for the purified parDMFase were carried out using the commercially available

Synchrotron X-ray fluorescence spectroscopy (syncXRF)
The identity of the metal present in parDMFase was validated by XRF of protein crystals. Protein crystals were flash frozen in liquid N2, and sequentially washed four times in the cryoprotectant consisting of 9:1 v/v of mother liquor and ethylene glycol. XRF scans were taken at the PROXIMA-1 beamline at the SOLEIL synchrotron at 107K. The spectrum contains X-ray emission lines characteristic of Fe. The peak at ~7,100 eV corresponds to the Kα emission energy of iron.

Electron Paramagnetic Resonance (EPR) study of parDMFase
Protein samples for EPR were prepared by concentrating the protein up to 20 mM in buffer A.
Samples were taken in the probe and frozen in liquid nitrogen. EPR spectra were obtained on a Bruker EMX EPR spectrometer at IIT Kanpur. Spectra processing and simulation were performed using a Bruker WIN-EPR and SimFonia software. A control experiment with buffer was also carried out.

Salt, temperature, and solvent dependent parDMFase stability and activity
To understand the effect of temperature and salt concentration on the activity and stability of parDMFase, we performed steady-state enzyme assays, and thermal shift assays in various assay conditions. All experiments were performed in sodium phosphate buffer (25 mM, pH 7.2, NaCl 50 mM, DTT 1 mM) and in triplicate. For thermal shift measurements, enzyme at different salt concentrations, parDMFase (600 nM) were first incubated for 24 hours at room temperature in phosphate buffer containing increasing NaCl concentrations ranging from 50-5000 mM. the inflection temperature (Ti) of each sample was obtained by thermal shift assay performed on a Tycho NT.6 system. Next, we performed steady-state enzyme kinetics experiments to determine the relative activity of parDMFase towards DMF. The measurements were carried out in 50 L reaction mixture, containing final concentrations of enzyme (600 nM) and DMF (300 mM) at 37°C for 20 min. Enzymatic reaction was inhibited by protein denaturation by adding TCA, and further a colorimetric assay was performed to determine the DMA concentration as described above.
Similarly, the effect of temperature on enzyme activity was measured by performing steady-state enzyme assay at different temperatures and measuring the relative catalytic activity with reference to the activity at 37°C.
To understand the solvent-based stability of parDMFase, parDMFase was incubated for at least 24h in different DMF-buffer concentrations at 4°C. The structural integrity of parDMFase was measured by thermal unfolding profile using a Tycho 1.6. The enzymatic response of incubates was recorded by a time-independent assay at 37°C. The thermal unfolding characterization of incubates shows a gradual decay in mean Ti with an increased organic-aqueous ratio. Interestingly, DMF showed a stabilization effect (with increased Ti of 3°C) up to ~7.5% v/v solvent content. A higher organic --aqueous ratio leads to conformational instability by monitoring the blue shifts in the detected first inflection temperature. The loss of the native thermal unfolding profile occurs after 65% v/v solvent presence (>7M). Enzyme catalytic response showed gradual decay of activity as opposed to a sharp decline in higher solvent medium. Optimum relative activity measured for DMF was at 3.1% v/v. In high organic solvent medium (>40%), disintegration of the catalytic structure leads to the loss of total activity even with the remaining residual quaternary structure.

Density functional theory calculations for strained iron in parDMFase
We used a recently published method for calculating the electronic energies of the iron complex using density functional theory [12] with the B3LYP functional for singlet calculations, the UB3LYP method for doublet calculations, and the 6-311G(d) basis set. We used the 6-311G(d) basis set for all atoms, as the difference in computation time was not significant. We assumed a low spin state of iron in both octahedral and tetrahedral calculations. The Fe 3+ charge state results in a doublet, and the Fe 2+ charge state results in a singlet regardless of the tetrahedral or octahedral symmetry around the iron atom. Moreover, both tyrosine residues were assumed to be deprotonated, along with the glutamic acid residue. Therefore, the Fe 3+ case results in a neutral system, whereas the Fe 2+ case results in an anionic state. The initial coordinates were obtained from the crystal structure and the beta carbon was replaced with a methyl group to sever as a single bond between two SP 3 carbons. For each geometry and charge state, two optimizations were performed: the first was constrained so that only the hydrogens and water atoms were allowed to move (strained state), and a second optimization was were all atoms were allowed to move (relaxed state). For the doublet calculations, where an unrestricted calculation was performed, the <S 2 > values were calculated before and after the annihilation of the first spin contaminant (Supporting information Table 3).
The expected value of <S 2 > for the system lacking any spin contamination is 0.75 for the doublet system. Since <S 2 > after spin annihilation for both Fe 3+ symmetries are 0.7559 and 0.7503 (Supporting information Table 3), which is relatively close to 0.75, we conclude that our calculations do not suffer from spin contamination. All calculations were performed using  [13] or Chimera [14] .