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New Mechanistic Insight from Substrate and Product Bound Structures of the Metal-dependent Dimethylsulfoniopropionate Lyase DddQ Adam E. Brummett, and Mishtu Dey Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00585 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 21, 2016

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Biochemistry

New Mechanistic Insight from Substrate and Product Bound Structures of the Metal-dependent Dimethylsulfoniopropionate Lyase DddQ$

Adam E. Brummett and Mishtu Dey* Department of Chemistry, The University of Iowa, Iowa City, IA 52242.

Running Title: Tyr120 is responsible for DddQ catalyzed β-elimination of DMSP

*To whom correspondence should be addressed. Mishtu Dey, Department of Chemistry, University of Iowa, W285 Chemistry Building, Iowa City, IA 52242-1727. Tel: 319-3841319. Fax: 319-335-1270. E-mail: [email protected].

$

This work was supported by University of Iowa College of Liberal Arts and Sciences.

The authors declare no conflict of interest.

X-ray crystallography, atomic coordinates, and structure factors of have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5JSP, 5JSR, and 5JSO).

ACS Paragon Plus Environment

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Abbreviations: DMSP, dimethylsulfoniopropionate; DMS, dimethylsulfide; Tris, 2-amino-2hydroxymethyl-propane-1,3-diol;

MES,

polyethylene

Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane;

glycol;

Bis-Tris,

2-(N-morpholino)ethanesulfonic

acid;

PEG,

DSBH, double-stranded beta-helix.

Keywords:

Dimethylsulfoniopropionate,

dimethylsulfide,

acrylate,

lyases,

cupin,

metalloenzyme.


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Biochemistry

Abstract The marine microbial catabolism of dimethylsulfoniopropionate (DMSP) by the lyase pathway liberates ~300 million tons of dimethylsulfide (DMS) per year, which plays a major role in the biogeochemical cycling of sulfur. Recent biochemical and structural studies of some DMSP lyases, including DddQ reveal the importance of divalent transition metal ions in assisting DMSP cleavage. While DddQ is believed to be zinc-dependent primarily based on structural studies, excess zinc inhibits the enzyme. We examine the importance of iron in regulating the DMSP β-elimination reaction catalyzed by DddQ as our as-isolated purplecolored enzyme possesses ~0.5 Fe/subunit. The UV-visible spectrum exhibited a feature at 550 nm, consistent with tyrosinate-Fe(III) ligand-to-metal charge transfer transition. Incubation of as-isolated DddQ with added iron increases the intensity of 550 nm peak, whereas addition of dithionite causes a bleaching as Fe(III) is reduced. Both the Fe(III)oxidized and Fe(II)-reduced species are active, with similar kcat values and 2-fold differences in Km for DMSP. The slow turnover of Fe(III)-bound DddQ allowed us to capture a substrate bound form of the enzyme. Our DMSP-Fe(III)-DddQ structure reveals conformational changes associated with substrate binding and shows that DMSP is positioned optimally to bind iron and is within proximity of Tyr 120 that acts as a Lewis base to initiate catalysis. The structures of TRIS-, DMSP-, and acrylate-bound forms of Fe(III)-DddQ reported here illustrate various states of the enzyme along the reaction pathway. These results provide new insights into DMSP lyase catalysis and have broader significance for understanding the mechanism of oceanic DMS production.


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Globally, a billion tons of the sulfur-containing molecule dimethylsulfoniopropionate (DMSP), is made each year

1, 2

DMSP is the main precursor responsible for liberating the

most abundant biogenic sulfur gas, dimethyl sulfide (DMS) emitted to the atmosphere.3 Approximately 300 million tons of DMS are generated per annum

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and this molecule is

implicated in climate regulation. DMSP can occur at remarkably high intracellular concentration (~ 0.5 M) in many marine phytoplankton such as macroalgae, diatoms, dinoflagellates, coccolithophores, a few angiosperms, and corals.1, 2, 5 Many of these marine eukaryotes, e.g. Emiliania huxleyi, produce DMSP as an osmoprotectant.6 The vast majority of DMSP liberated into the marine environment is degraded by these microorganisms through either the demethylation or lyase pathways (Figure S1) and supplies 3-10% of the carbon and 30-100% of their sulfur.4, 7 A highly abundant marine αproteobacterium of SAR11 species is believed to degrade DMSP directly by the demethylating enzyme, DmdA.8,

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The demethylation pathway of DMSP biodegradation

generates methylmercaptopropionate and major downstream catabolites, methanethiol, and acetaldehyde.10 The lyase pathways of DMSP biodegradation exist in a wide range of organisms, including bacteria, eukaryotic phytoplankton, macroalgae, and fungi. Microbial DMSP cleavage produces the volatile DMS and the catabolites, acrylate or 3hydroxypropionate (3HP) through elimination reactions (Figure S1).4, 11 Regardless of the microbe, DMS generated by the lyase pathway serves as a key nutrient for marine bacteria,11 a chemo-attractant for zooplankton and seabirds,12-15 has wide industrial uses,16 and more importantly it is thought to influence global climate.3 DMS emitted to the atmosphere is oxidized forming acidic sulfate aerosols that act as cloud condensation nuclei and reflect sunlight, thus affecting climate. In turn, these products are delivered back to Earth as acid rain, representing a key component of the global sulfur cycle.11, 17 In addition to DMS, the other DMSP cleavage product acrylate is a compound of high industrial value.


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Biochemistry

Due to the environmental and industrial significance of DMSP, recent genetic analyses led to the identification of the genes and enzymes involved in DMS and acrylate productions.4 However, very little is known about the DMSP lyases at a biochemical level. To date, six distinct DMSP lyases occur in bacteria and fungi, which are encoded by various ddd genes4 and belong to different protein families (Figure S1). The class III CoA-transferase DddD, also exhibiting lyase activity, forms 3HP and DMS as major products when grown with DMSP and acetyl-CoA (Figure S1).18-21 In contrast, the other known enzymes (DddY, DddP, DddQ, DddL, DddW) cleave DMSP to produce acrylate and DMS (Figure S1).4 The DMSP lyase DddY is a periplasmic protein and has no known conserved protein domains,22 but DddP belongs to the M24B metallopeptidase family and consists of a diiron center.23-26 The other lyases DddL, DddQ, and DddW belong to the cupin superfamily of enzymes, which is characterized by a double stranded β-helical (DSBH) structure

27-29

consisting of

eight anti-parallel β-strands and contains the conserved active site residues (Figure 1). These enzymes are characterized by 2/3 His and 1-carboxylate (Asp/Glu) residues at the active site that coordinate various divalent transition metal ions.27-30 A vast majority of proteins containing the cupin fold require Fe(II) for catalytic activity.27-30 Based on biochemical studies, the DMSP lyase DddW from Ruegeria pomeroyi DSS-3 has been shown to be promiscuous in metal recognition.31 While DddW preferentially uptakes iron from a mixture of metal ions, the DMSP cleavage activity is dependent on both Fe(II) and Mn(II).31 Structural studies on the DMSP lyase DddQ from Ruegeria lacuscaerulensis displayed a zinc ion at its active site.32 While DddQ is proposed to be a zinc-dependent enzyme as it was isolated with 42% zinc based on atomic absorption spectral analysis, activity measurements indicate that excess zinc inhibits the DMSP cleavage activity of DddQ. In contrast, excess Co(II) and Mn(II) resulted in an increase in the enzyme activity.


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Interestingly, no biochemical data is available examining the effect of iron and nickel on DddQ activity. The metallocofactor specificity of the DMSP lyases is unclear and the mechanism of DMSP cleavage to DMS and acrylate is not fully established. One mechanism, based on structural, mutational, and biochemical analyses of DddP, includes an ion-shift mechanism involving a diiron active site 26. In a second mechanism, based on site-directed mutagenesis and metal uptake studies of DddW, a Fe(II) cofactor assists in the catalytic reaction and the proton abstraction from the β-carbon of DMSP is proposed to be initiated by a histidine, although the involvement of a tyrosine was not ruled out.31 A third mechanism, based on crystal structures of the inactive Zn-DddQ, invokes tyrosine acting as a base to abstract a proton from DMSP resulting in β-elimination of acrylate and DMS.32 The structure of an active DMSP lyase containing the DSBH/cupin fold has not been determined. In order to gain insight into the mechanism of DMSP cleavage reaction catalyzed by DddQ, it is important to understand how the active site residues are organized to assemble an active metallocofactor for substrate positioning and catalysis. In the present study, we examine the binding of iron, substrate, and product to DddQ using X-ray crystallography as the primary tool. We have identified key catalytic residues and observed that DddQ is active in presence of both Fe(II) and Fe(III). Furthermore, the crystal structures reveal conformational changes upon substrate and product binding, and represent the ‘resting’, ‘catalytic’, and ‘product’ states of the enzyme in the catalytic cycle proposed here. Based on the structural studies, we propose a new mechanism of DMSP cleavage catalyzed by DddQ.

Materials and Methods


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Protein

Expression and

Purification. The gene encoding DddQ from R.

lacuscaerulensis ITI_1157 in the pET22b vector was a generous gift from Yu-Zhong Zhang. The plasmid DNA was transformed into E. coli BL21 (DE3) (Life Technologies, Grand Island, NY) for protein expression. The enzyme was isolated as previously reported with slight modification to the anion exchange chromatography.32 To summarize, 12.3 g of cell paste was suspended in 50 mM Tris-HCl pH 8.0, 200 mM NaCl (lysis buffer), lysed, spun down and the supernatant was loaded onto a nickel-nitriloacetic acid column. After wash steps, DddQ protein was eluted with the lysis buffer containing 50 mM imidazole. The eluted fraction was dialyzed in the lysis buffer to remove imidazole. The dialyzed protein was loaded onto a 10 mL Q-sepharose column, where DddQ came in the flow through, but impurities eluted at higher salt concentrations. DddQ containing fractions were concentrated to 2 mL using Amicon Ultra 5,000 molecular weight cut-off membrane (Millipore, Billerica, MA) and loaded onto a Superdex 200 16/60 gel filtration column (120 mL, GE Healthcare) pre-equilibrated with 10 mM Tris-HCl pH 8.0, 100 mM NaCl (storage buffer). The eluted fractions were collected, purity checked, protein concentrated to 5.8-7.0 mg/mL, and individual aliquots were stored at -80 °C until use. Reconstitution of DddQ. The as-isolated R. lacuscaerulensis DddQ was reconstituted by adding freshly prepared solution of 100 mM (NH4)2Fe(SO4)2 to a final concentration of 0.5 mM and incubated for 1 min. Unbound iron was removed by sequential dialysis of the protein samples in a chelation buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA) for 12 hours followed by dialysis in a non-EDTA containing buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl) for 18 hours. Since the reconstitution was performed aerobically, unless otherwise specified, all iron present in holo-DddQ is in the oxidized Fe(III) state. The iron content of as-isolated and reconstituted holo-DddQ was confirmed by inductively coupled plasma optical emission spectroscopy (ICP-OES) and the ferrozine assay.33


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Determination of iron content by ferrozine assay and ICP-OES. The iron content of DddQ was analyzed with samples of as-isolated and reconstituted holo-forms of the enzyme. The assay mixture consists of 30 µL enzyme sample mixed with 70 µL of 2 M HCl in an Eppendorf tube and boiled for 5 min at 95 °C. The denatured enzyme solution was then centrifuged at 13,400 rpm for 12 min at room temperature. The supernatant was removed and kept in another Eppendorf tube, while the pellet was re-suspended in 100 µL of 2 M HCl. The resuspension was boiled at 95 °C for 15 mins and then centrifuged at 13,400 rpm for 12 mins at room temperature. The second supernatant was combined with the first, and to that the following was added: 200 µL of 10 mM ferrozine, 40 µL of 75 mM sodium ascorbate, 200 µL of a saturated ammonium acetate solution (~19 M), and 360 µL of water for a final volume of 1 mL. The ferrozine and sodium ascorbate solutions were always prepared fresh. The absorbance was measured at a wavelength of 562 nm in a 1 cm cuvette for the solution (extinction coefficient: 27.9 mM-1cm-1). The concentration of iron present was determined for the 1 mL solution and the dilution factor was used to calculate the concentration of iron present in the original 30 µL enzyme solution to get a ratio of iron to enzyme. DddQ samples were analyzed by ICP-OES in the as-isolated and reconstituted-holo forms. For each sample, 200 µL of each enzyme was denatured with 40 µL of concentrated nitric acid and diluted to 2 mL with water. The denatured enzyme was removed by centrifugation at 4,000 rpm for 15 mins. The supernatant was analyzed for content of the following metals: Mn, Fe, Co, Ni, Cu, and Zn and compared against a blank containing buffer, nitric acid, and water of the same composition as the samples. UV-visible spectroscopy. The UV-visible spectra of DddQ were measured for asisolated, reconstituted Fe(III)-holo, and reduced-Fe(II) protein samples. All spectral measurements were performed in an anaerobic environment with an Ocean Optics DH 2000BAL light source (Ocean Optics, Dunedin, FL). Spectra were measured between 200-900 nm


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Biochemistry

in a 100 µL buffer containing 50 mM Tris pH 7.4, 30 mM NaCl at 290 µM protein concentration. The spectrum of an as-isolated DddQ sample was measured first, followed by a reconstituted holo-Fe(III) spectrum. The reduced Fe(II)-DddQ sample was prepared by adding100 mM sodium dithionite solution to holo-Fe(III) at a final concentration of 2 mM. DMSP Lyase Activity Assays. Activity assays were performed in the presence of various divalent transition metal ions with 2 µM apo-DddQ, 15 mM DMSP, and 250 µM respective metal ion. To determine the kinetics parameters in presence of iron, steady state assays were performed using 20 µM reconstituted-Fe(III)-DddQ in 50 mM HEPES pH 8.0, 100 mM NaCl at 30 °C. The kinetic parameters in the presence of Fe(NH4)2(SO4)2 were determined by performing the activity assays anaerobically using a 100 mM stock solution of sodium dithionite with a final concentration of 2 mM. The substrate, DMSP, was added to the enzyme reaction solution with final concentrations ranging from 0.2 mM to 200 mM in a 700 µL reaction volume. Aliquots of 100 µL were taken at various time points over a 4 min range to monitor product (acrylate) formation. Samples were analyzed following protocols as previously reported with DddW.31 Crystallizations. All DddQ crystals were obtained using the sitting-drop vapor-diffusion method at 20 °C from the PACT screen (Molecular Dimensions) by mixing 1 µL of asisolated protein with 1 µL of precipitant solution. Initial crystals of DddQ appeared as stacked plates in 0.2 M NaI, 20% PEG 3350 with 5.75 mg/mL protein. Optimization of the condition did not produce single crystals. However, these crystals were used as micro-seeds (1:100 dilution of seed stock) in subsequent crystallization experiments with sparse matrix and optimized screens. Both Fe(III)-DddQ-DMSP and Fe(III)-DddQ-acrylate bound structures were obtained by optimization of a condition observed from seeding the PACT coarse screen.


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Crystallization of Fe(III)-DddQ-DMSP. A solution of 7 mg/mL as-isolated DddQ was mixed with 10 mM DMSP and crystal drops were set by mixing 1 µL of this solution with equal volume of reservoir solution of the PACT screen, followed by the addition of 0.2 µL of seed stock. Initial crystals appeared in 1-2 days and were optimized by varying pH and precipitant concentration. Diffraction quality crystals were obtained from 0.1 M Bis-Tris propane pH 6.5, 0.2 M NaBr, 21% PEG 3350. The crystals of Fe-DddQ-DMSP were plate shaped and grew in one day. The crystals were soaked for 1 minute in a cryoprotectant solution of 0.1 M Bis-Tris propane pH 6.5, 0.2 M NaBr, 25% PEG 3350, 10 mM DMSP, 20% glycerol and frozen in liquid nitrogen. Crystallization of Fe(III)-DddQ-acrylate. Crystals of Fe(III)-DddQ bound with acrylate were obtained from the PACT screen using a solution of 7 mg/mL as-isolated DddQ and 1.5 mM acrylate incubated on ice for 10 minutes. Crystal drops were set with 1 µL of the above protein solution mixed with 1 µL of precipitant solution and 0.2 µL of seed stock. Crystals were obtained in 0.1 M Bis-Tris propane pH 6.5, 0.2 M NaBr, 23% PEG 3350 and grew after a day. The crystals were soaked briefly in a cryoprotectant solution containing 0.1 M Bis-Tris propane pH 6.5, 0.2 M NaBr, 25% PEG 3350, 1.5 mM acrylate, 20% glycerol before submersion in liquid nitrogen. Crystallization of Fe(III)-DddQ-TRIS. Crystals of the TRIS bound form of Fe(III)DddQ were obtained from the PACT screen using a 1:100 dilution of seed stock. Diffraction quality crystals were obtained by mixing 1 µL of 5.8 mg/mL as-isolated DddQ with 1 µL of precipitant solution (0.1 M MES pH 6.0, 0.2 M LiCl, 20% PEG 6000) and 0.2 µL of seed stock. The crystals were submerged in a cryoprotectant solution containing 0.1 M MES pH 6.0, 0.2 M LiCl, 22% PEG 6000, 20% glycerol, prior to cooling in liquid nitrogen. X-ray Data Collection and Analysis. Data sets for DMSP- and acrylate- bound Fe(III)DddQ were collected at 100 K at the Advance Photon Source, beamline 19-BM. The data


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collection parameters were: Fe(III)-DddQ-DMSP, 0.5° oscillation, 0.5 s exposure time, 183 mm detector distance; and Fe(III)-DddQ-acrylate, 0.5° oscillation, 0.5 s exposure time, 195 mm detector distance. The DddQ-TRIS bound data set was collected at 100 K at the Advanced Light Source, beamline 4.2.2, using 0.2° oscillation, 0.3 s exposure time, and 210 mm detector distance. All data sets were indexed and integrated using the XDS package34 and merged and scaled using SCALA35 (CCP4 suite).36 Data collection and refinement statistics are summarized in Table 1. Structure Determination. For all data sets, molecular replacement was performed with PHASER (CCP4 suite)37 using a monomer of R. lacuscaerulensis DddQ (PDB ID 4LA2) as a search model while looking for 2 copies. Refinement was carried out using phenix.refine from the PHENIX software package.38 Since all structures contain a coordinated metal ion, bond length and angle parameters were generated by phenix.metal_coordination. All chains in each structure contain a bound iron at the active site. The C-terminus end in all chains of the DddQ structures, have only 10 disordered residues (8 of which are due to 6x-His tag and linker). Although there is electron density indicating the presence of flexible amino acids, the density quality is too poor to model. The disordered residues that were not modeled for each structure and chains are 191-200. The N-terminus end of DddQ has an extra methionine that exists as a cloning artifact and is only ordered enough to model in chain A of the Fe(III)DddQ-DMSP structure. Following repeated rounds of refinement using simulated annealing, energy minimization, real space refinement, B-factor refinement, rigid-body refinement, and occupancy refinement, model building was done using COOT.39 Addition of ligands into distinct positive electron density was based on a simulated annealing omit map generated by omitting the ligand. Water molecules were added into clear densities in later rounds of refinement. At later stages of refinement, simulated annealing, rigid-body and real space refinement were turned off and only B-factor refinement was used. Composite omit maps


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were generated to verify the final models. The structures were validated with PROCHECK,40, 41

the wwPDB validation server,42 and MOLPROBITY.43 The values in Table S1 are from

PROCHECK and MOLPROBITY. CAVER 3.0.1 was used for the analysis and visualization of cavities and tunnels. 44 All structural figures were made using PyMOL.

Results

Spectral and kinetic characterization of iron-bound DddQ. Based on the amino acid sequence and the reported crystal structures

32

, DddQ contains conserved cupin motifs and

residues known to bind metal ions (Figure 1). The reported biochemical characterization of R. lacuscaerulensis DddQ indicates that zinc, copper, and alkaline earth metal ions inhibit the enzyme, whereas Co(II) and Mn(II) enhance DMSP cleavage activity of DddQ

32

similar to

that observed for DddW.31 However, the previous study did not examine the enzyme activity in the presence of iron. Our metal content analysis indicates that as-isolated DddQ consistently contained 0.45-0.6 equivalents of bound iron (~50%), whereas, very little zinc (

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