Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.


Expression of full-length NcCAR wt and cleavage to subdomains
The expression of Neurospora crassa CAR (Q7RW48) was conducted in Escherichia coli BL21 Star TM (DE3) cells using the pETDuet1 vector as previously described by Schwendenwein et al. with EcPPTase (CAQ31055.1) at multiple cloning site (MCS) 1 and the NcCAR sequence in MCS2 (NcCAR wt , Table S1). 1 Variant NcCAR Y844A was cloned and produced as described previously. 2 To facilitate the purification of individual domains, a WELQut protease cleavage site was introduced after residue 648 to create the construct AT-WELQ-648-R. After size exclusion chromatography, the construct was diluted to a concentration of 1 mg/mL in Tris/HCl-buffer (0.1 M, pH 8.0). 1 µL of WELQut protease (5 U/µL Thermo Fischer Scientific Inc., Waltham, USA) was used per 0.53 µg of cleavable protein. The digestion was performed for 26.5 h at 16 °C and 500 rpm. Subsequently, the domains were separated via size exclusion chromatography and used for crystallography. 3. Cloning and Expression of the R-domain of NcCAR (NcCAR Δ1-649 ) and control strains A gene for the construct corresponding to the cleavage product of the AT-WELQ-648-R was cloned with an N-terminal His-tag and a TEV-protease cleavage site by addition of MSYYHHHHHHHHHHDYDIPTTENLYFQGA to the respective subunit. The resulting vector designated pETDuet:EcPPTase_NcCAR_R_domain_Nterm_10xHisTag was used for transformation of electrocompetent E. coli BL21 Star TM (DE3) and the gene was expressed as described above. 6.0 g cells were lysed by sonication in 40 mL sodium phosphate buffer (20 mM, pH 7.4) containing 500 mM NaCl and 10 mM imidazole. Cell debris was removed by centrifugation using an Avanti J-26-XP centrifuge (Beckman Coulter, Brea, USA) equipped with a JA 25.50 rotor (Beckman Coulter, Brea, USA) for 30 minutes at 4 °C at 16,000 rpm. The supernatant was filtered through a 0.45 µM syringe filter and applied to a 5 mL HisTrap Fast Flow column (GE Healthcare, Chicago, USA) using an ÄKTA system (Cytivia, Marlborough, USA) and eluted with sodium phosphate buffer (20 mM, pH 7.4) containing 500 mM NaCl and 500 mM imidazole. R-domain containing fractions were concentrated to a final volume of 28 mL and a concentration of 3 mg/mL. 400 µL TEV protease (kindly provided by Tamara Berger, 1.5 mg/mL) and 21 mg DTT were added and incubated for 3 h at 22 °C. The cleaved protein was dialyzed in 5 L of sodium phosphate buffer (20 mM, pH 7.4) containing 500 mM NaCl, 10 mM imidazole and 1 mM DTT overnight. The dialyzed protein solution was loaded to a Ni-NTA column and treated as described above. The cleaved R-domain, corresponding to NcCAR Δ1-649 with two additional N-terminal residues GA was eluted using sodium phosphate buffer (20 mM, pH 7.4) containing 500 mM NaCl, concentrated and loaded to a HiLoad ® 16/600 Superdex 200 pg size exclusion column (GE Healthcare, Chicago, USA) and eluted with Tris/HCl buffer (10 mM, pH 7.4) containing 150 mM NaCl at a flow rate of 0.5 mL/min. The separately expressed R-domain was used for crystallization trials and activity assays. Similarly, the single A domain of NcCAR was cloned and produced (NcCAR Δ550-1052 ) as a control (Table S1). The solvent content was estimated based on the calculated Matthews coefficient. 4 Phasing was performed using a truncated ensemble made from distant homologues employing the CCP4mg-MrBUMP task. 5 Phases were improved by SHELXE 6 to produce a c-alpha trace followed by model building employing Buccaneer. 7 Refinement was conducted by repetitive rounds of REFMAC in CCP4 and manual model building in COOT. 4,8 A model has been deployed with PDB-code 8AEP.
SAXS data were collected on the BioSAXS BM29 beamline (ESRF, Grenoble) equipped with the Pilatus3 2M detector (Dectris, Baden-Dättwil, Switzerland) and a sample-to-detector distance of 2.867 m. An X-ray wavelength of 0.99 Å (12.5 keV) was used for the measurement. Data were collected on 3 concentrations ranging from 1 to 5 mg/ml NcCAR-R in with 10 mM Tris/HCl buffer pH 7.4 containing 150 mM NaCl. Buffer measurements were performed between each sample. For each data collection, 20 frames of 100 ms were collected. Scattering collected on the pure buffer was subtracted from that of the sample, and the resulting curves were normalized to the protein concentration.
The data processing was performed within the ATSAS package. 9 The program CRYSOL was used to compute the theoretical curves from the atomic structures. 10 Volume fractions of the components of the oligomeric mixtures (monomer + crystallographic dimer) were computed and fitted to the data using the program OLIGOMER. 11   Figure S3). The adjacent helix is involved in the formation of the tunnel that is putatively harboring the phosphopantetheine moiety (compare Figure 3 and 5).

Determination of oligomerization state of full length NcCAR
NcCAR expressed similarly to methods described in Section 3. NcCAR was purified via IMAC with a 5 mL HisTrap Fast Flow column (GE Healthcare, Chicago, USA) using an ÄKTA system (GE Healthcare, Chicago, USA) and eluted with Tris/HCl buffer (20 mM, pH 7.4) containing 500 mM NaCl and 500 mM imidazole. Eluted NcCAR was concentrated to 0.5 mL and loaded onto a HiLoad ® 16/600 Superdex 200 pg size exclusion column (GE Healthcare, Chicago, USA) and eluted with Tris/HCl buffer (20 mM, pH 7.4) containing 150 mM NaCl at a flow rate of 0.5 mL/min. Gel Filtration Standards (Bio-Rad, USA) were reconstituted in 0.5 mL deionized water, loaded and eluted as aforementioned ( Figure S5). The molecular mass of the main peak eluting at 69 mL ( Figure S5A) was determined to be 116 kDa, which corresponds to NcCAR in its monomeric form. A peak eluting at approximately 58 mL retention volume is also comprised of NcCAR according to gel electrophoreses and likely represents a trimer.

Figure S5. Size exclusion chromatography of A) full length NcCAR and B) Gel Filtration Standards (BioRad) C) SDS-PAGE profiles for the purification of full length NcCAR. STD: Prestained Protein Standard (New England
Biolabs), 1: peak eluted at 58 mL and 2: peak eluted at 69 mL.

Synthesis of thioester library
The synthesis of the respective thioesters was described previously. 13 They are summarized in Table S3, with the corresponding GC-FID method (SI, Chapter 6).

Biotransformations
Biotransformations were carried out in triplicates using cell free extracts (CFE) as well as purified enzymes. Cell free extracts were diluted 1:10 (~10mg/mL) in 50 mM sodium phosphate pH 7.75 in a volume of 1 mL per reaction. Purified enzymes were thawed on ice and diluted to appropriate concentrations (8-9 µM) in 50 mM sodium phosphate pH 7.75. Reactions were initiated with 1 % DMSO and 10 mM NADPH at 30 °C for approximately 20 hours. After selected times, reactions were terminated with the addition of HCl (6 M). Subsequently, 500 µL of each bioconversion was vortexed for 2 min after addition of equal volumes of ethyl acetate to extract organic compounds. The organic phase was dried with Na2SO4 (approximately 10 % (w/v)) prior to transferring 200 µL into glass vials for GC analysis.
A Shimadzu 2010 Plus equipped with a flame ionization detector and a ZB-5 column (30 m, 0.25 µm, 0.32 mm, Agilent technologies) was used to analyze the reactions. Sample aliquots of 1 μL were injected in split mode (split ratio 10:1). Methods for each substrate group was adapted in order to reach clear separation of substrate and reduced product. Analytical standards, as well as internal standard tetradecane (0.01 %), were used to determine analyte concentrations (Table S4).

Reduction of 1a & 4a with CFE
We wondered, whether typical short chain dehydrogenase substrates would be reduced; hence, we subjected the CFE from E. coli ( Figure S6) clones expressing the R-domain, or the Adomain or the full-length Y844A variant (the latter two as controls) to the aromatic aldehyde 2a and the simple aromatic ketone 5b. Cofactor recycling, adapted from Strohmeier et. al., was applied to provide enough NADPH. 14 Samples were analyzed by GC-FID (Table S5).

Product GC-Method 2a
Benzaldehyde  NcCAR R-domain reduced acetophenone 5b to its corresponding alcohol 6b. After 20 hours we observed more than 72% conversion. Aldehydes are actually considered as final products upon CAR reactions, so we decided to investigate putative over reduction, which are reported frequently. Reduction of 2a to 3a by the R-domain was confirmed by GC-FID analysis, but not surprisingly, this product was also found in the cell free-extracts of our controls. To gain a better understanding, we must go one step further and purify our constructs to reduce E. coli background activities.

Reductions with purified enzymes
We used immobilized metal-affinity chromatography (on Ni-Sepharose) to eliminate E. coli background activities from the NcCAR R-domain, the full-length NcCAR wt , and NcCAR Y844A variant as a negative control. Buffer exchange was performed via size exclusion chromatography and approximately 3 µg of protein was applied on each lane of the SDSpolyacrylamide gel slab ( Figure S7). Ketone reduction was confirmed with purified R-domain for 5a, 5b and 5c ( Figure S8). Here, the R-domain produced approximately 18% of 6b, and over 10% of both, 6a and 6c. Equimolar concentration of full-length enzyme showed much lower rate of conversions (8%) and further, theY844A variant did not reveal any conversions at all. To our knowledge, this is the first time that these 3 ketones have been reduced by a single domain of a fungal CAR.

Phylogeny
A set of sequences was retrieved from the NCBI and aligned with ClustalO. The respective alignment can be retrieved as Supplemental file S1. In Jalview a neighbor joining tree (NJ) was created that is depicted in Figure S10. Figure S10. Neighbor joining tree of carboxylate reductases. Four different clades can be identified, and the respective sequence names are highlighted in purple, green, blue and red, respectively. Grouping of nonhighlighted sequences is out of the scope of this work.
ConSurf server cited AlphaFold and ConSurf: Identification of Functional Regions in Proteins by Surface-Mapping of Phylogenetic Information ( Figure S11). Figure S11. Representation of highly conserved residues in the overall topology of full-length NcCAR. Residues that are conserved more than 90 % are depicted as spheres (color gradient: green 90 % conservation to blue 100 %). The coloring of the secondary elements is according to the respective subunits.