Cofactor Specificity Switch on Peach Glucitol Dehydrogenase

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Cofactor specificity switch on peach glucitol dehydrogenase Matías D. Hartman, Romina Inés Minen, Alberto A. Iglesias, and Carlos M. Figueroa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01240 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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Biochemistry

Cofactor specificity switch on peach glucitol dehydrogenase Matías D. Hartman, Romina I. Minen, Alberto A. Iglesias and Carlos M. Figueroa* Instituto de Agrobiotecnología del Litoral, UNL, CONICET, FBCB, 3000 Santa Fe, Argentina

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ABSTRACT

Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than for NADP+. Herein, we combined structural and kinetic data to reverse cofactor specificity of this enzyme. Using site-saturation mutagenesis we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.


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Biochemistry

INTRODUCTION Sugar-alcohols (alditols) are extensively used in food and pharmaceutical industries, as they have several advantages over regular sugars (aldoses and ketoses), including: i) low physiological calorific value, ii) insulin-independent signaling metabolism, iii) non-cariogenic activity, and iv) reduced chemical reactivity.1 Alditols are also used for personal care items, animal nutrition, and production of chemicals, such as propylene glycol, synthetic plasticizers and alkyd resins.2 Sugar-alcohols naturally occur in many plants and microorganisms, where they play key roles, such as carbon storage and transport, free-radical scavenging, and osmoprotection.3 Most alditols are produced on a commercial scale by catalytic reduction of sugars with hydrogen in the presence of Raney nickel at high temperature and pressure,1 while microbialbased processes are receiving increasing attention.4 Reduction with hydrogen is very expensive in terms of energy consumption and releases toxic by-products, whereas microbial production requires downstream detoxification procedures and product separation from the fermentation broth.1,4 The use of enzyme-based processes for production of sugar-alcohols offer three main advantages: i) catalysis is performed at low pressure and temperature; ii) chemo-, regio- and stereo-selectivity; and iii) no microorganisms are involved in the process (besides those used for enzyme preparation).5 However, the high costs associated with enzyme production and cofactor regeneration have made such strategies less appealing for industrial purposes. Besides that, the use of formate dehydrogenase, which produces CO2 from formate with the concomitant reduction of NAD+, has been patented for the industrial-scale production of L-trimethyl leucine.6


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Enzyme cofactors (in particular, NAD+ and NADP+) are extremely expensive to be used in stoichiometric amounts for large-scale processes. To circumvent this problem, a second enzymatic reaction is usually coupled to the reaction of interest to recycle the cofactor.7 The use of dehydrogenases (oxidoreductase superfamily, EC class 1) in biocatalytic processes and metabolic engineering has turned coenzyme switching into a top research area. Furthermore, enzymes capable of using both cofactors might be desirable for increasing the diversity of coupled reactions and the efficiency of particular processes.8 Oxidoreductases from the EC subclass 1.1 (which act on the CH-OH group of donor substrates) use a plethora of acceptor substrates, including oxygen, cytochromes, and dinucleotides.9 In general, dehydrogenases that bind NAD(H) or NADP(H) are highly specific for the coenzyme they use.10 Most oxidoreductases that bind these dinucleotides display a domain known as the Rossmann fold, which contains a βαβ subdomain that interacts with the ADP moiety of the dinucleotide (Figure 1).11 Two different sites within the βαβ fold are important for coenzyme binding and specificity. The consensus sequence of site 1 (located between the first β-strand and the α-helix) is GXGXX[G/A]; the last position varies between Gly and Ala in NAD- and NADP-binding enzymes, respectively.12–14 Site 2 is located at the end of the second β-strand and has different properties depending on the cofactor used: negatively charged amino acids that repel NADP(H) in NAD-binding enzymes15 or larger pockets with polar and positively charged residues in NADP-binding enzymes.8


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Biochemistry

Figure 1. Homology model of peach GolDHase in complex with NAD+. The figure shows a detailed view of the βαβ subdomain within the Rossmann fold. Amino acids from site 1 (1 to 6) are close to the phosphate groups linking both nucleosides. Residues from site 2 are close to the 2‟ and 3‟ hydroxyl groups of the adenosine moiety (red asterisk).

Glucitol (Gol; an alditol also known as sorbitol) is a main photosynthetic product in plants from the Rosaceae family.3 Gol dehydrogenase (GolDHase; EC 1.1.1.14) is a NAD-dependent enzyme that catalyzes the reversible oxidation of Gol to fructose.16–18 The enzymes from sheep and human liver display an ordered bi-bi mechanism, with the formation of a ternary complex, where the cofactor (NAD+ or NADH) binds first.19,20 Plant GolDHases are mainly found in heterotrophic tissues, such as developing leaves and fruits.3 We hypothesized that generation of NADPH (linked to Gol oxidation) in the cytosol of heterotrophic cells would be highly beneficial, particularly under abiotic stress conditions.21 To better characterize the structural domain determining cofactor specificity in plant GolDHases, we designed mutants to switch the


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capability of NAD(P)+ use. The triple mutant D216A/V217R/D218S, obtained by combining site-saturation and site-directed mutagenesis, effectively binds NADP+. Our results strongly suggest that the triple mutant has the potential of being used in metabolic and cellular engineering and for NAD(P)+ regeneration in industrial processes.

MATERIALS AND METHODS Bacterial strains and reagents Escherichia coli TOP10 cells (Invitrogen) were used for cloning procedures and plasmid maintenance and E. coli BL21 (DE3) cells (Invitrogen) were employed for protein expression. Gol, NAD+ and NADP+ used for enzyme activity assays were from Sigma-Aldrich. All the other reagents were from the highest quality available. Analysis of GolDHase sequences We retrieved 43 sequences of plant GolDHases from the Phytozome v9.1 server (https://phytozome.jgi.doe.gov/pz/portal.html) and from the NCBI database (http://www.ncbi.nlm.nih.gov/). Sequences were analyzed with the program BioEdit 7.0.5.322 and multiple alignment was performed with the Clustal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/). The WebLogo server (https://weblogo.berkeley.edu/logo.cgi) was used to determine consensus substrate binding motifs within plant GolDHases. Site-directed and site-saturation mutagenesis of GolDHase


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Biochemistry

The sequence coding for peach (Prunus persica) GolDHase (NCBI Protein ID BAA94084)18 was subcloned in the pRSFDuet vector between the BamHI and SalI sites. The construction [pRSFDuet/PpeGolDHase] was used to express the wild type (WT) enzyme and as a template for introducing mutations. We used the QuikChange Site-Directed Mutagenesis Kit (Agilent) to introduce mutations at cofactor binding sites 1 (G196A) and 2 (D216X and D216A/V217R/D218S). After selection of the D216A mutant (see below), we constructed the D216A/V217R/D218S triple mutant using the plasmid containing the D216A mutant as template. The primers and templates used to obtain mutations in both cofactor binding sites are described in Table S1. Site-saturation mutagenesis23 was used to obtain a library of mutants in the cofactor binding site 2 at position 216 (D216X). It has been described that 88 clones are enough to screen each of the 20 amino acids at least once.24 The library obtained by site-saturation mutagenesis was used to transform E. coli BL21 (DE3) cells and 100 clones were chosen to screen NADP-dependent GolDHase activity. Plasmids from positive clones were isolated using the Wizard Plus SV miniprep kit from Promega and sequenced by Macrogen (Seoul, Korea). Protein expression and purification E. coli BL21 (DE3) cells transformed with the WT and mutant versions of GolDHase were used to express the recombinant proteins. Cells were grown in 1 L of LB supplemented with 50 µg mL-1 kanamycin and 0.1 mM ZnCl2 at 37 °C and 200 rpm in an orbital shaker until OD600 was ~0.6 and then were induced with 0.2 mM isopropyl-β-D-1-thiogalactopyranoside at 25 °C overnight. Cells were harvested at 5000 x g and 4 °C and kept at -20 °C until use.


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Recombinant enzymes were purified using the following procedure. The cell paste was resuspended with 30 mL of Buffer H [25 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% (v/v) glycerol, 10 mM imidazole] and disrupted by sonication. The resulting suspension was centrifuged twice at 15000 x g and 4 °C for 15 min and the supernatant was loaded into a 1-mL HisTrap column (GE Healthcare) previously equilibrated with Buffer H and connected to an ÄKTA Explorer 100 purification system (GE Healthcare). The column was washed with 10 mL of Buffer H and proteins were eluted with a linear gradient of imidazole (10 to 300 mM, 50 mL). Fractions of 2 mL were collected and those containing the enzyme of interest were pooled, concentrated, and stored at -80 °C until use. Under these conditions, the proteins were stable for at least 3 months. Protein methods Proteins were electrophoresed under denaturing conditions (SDS-PAGE), as published by Laemmli.25 Enzyme purity was evaluated by densitometry using the software ImageJ 1.50i (https://imagej.nih.gov/ij/index.html). Protein concentration was determined with the Bradford reagent,26 using BSA as standard. The number of Zn2+ ions present in each subunit of the recombinant enzymes was determined using the Zincon reagent (Sigma-Aldrich), following the method developed by Säbel et al.27 Samples and ZnCl2 standards were prepared in borate buffer and proteins were denatured with 8 M urea. Production of antibodies and immunodetection Antibodies against peach GolDHase were raised in rabbits immunized with the purified WT recombinant protein (service provided by ICIVET, UNL-CONICET, Esperanza, Argentina). After SDS-PAGE, proteins were transferred to nitrocellulose using a wet-tank and transfer buffer


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Biochemistry

containing 20% (v/v) methanol at 180 mA for 1 h. Membranes were blocked with 5% (w/v) BSA in TBS supplemented with 0.05% (v/v) (TBST) for 1 h. Primary antibodies were diluted 1/1000 in 1% (w/v) BSA in TBST and membranes were incubated overnight at 4 °C. Membranes were washed in TBST and then incubated with anti-rabbit IgG conjugated to Alexa Fluor 647 (Invitrogen) diluted 1/5000 for 1 h. After washing in TBST, fluorescence was detected using a Typhoon 9400 scanner (GE Healthcare). Native molecular mass determination The native molecular mass of WT and mutant GolDHases was determined using a Superdex 200 10/300 column (GE Healthcare) equilibrated with 50 mM HEPES pH 8.0 and 100 mM NaCl. Standard proteins (ribonuclease, 14 kDa; carbonic anhydrase, 29 kDa; ovalbumin, 43 kDa; conalbumin, 75 kDa; aldolase, 158 kDa; ferritin, 440 kDa; and thyroglobulin, 669 kDa) were used to construct a calibration curve by plotting the Kav values versus log (molecular mass). Kav values were calculated as (Ve-V0)/(Vt-V0), where Ve is the elution volume of the protein, V0 is the elution volume of Dextran Blue and Vt is the total volume of the column. Enzyme activity assay and determination of apparent kinetic constants GolDHase activity was determined as described in Hartman et al.18 The standard assay mixture contained 100 mM Tris-HCl pH 9.0, 5 mM NAD+, 400 mM Gol, and the proper amount of enzyme. Alternatively, NAD+ was replaced by NADP+ (concentrations varied depending on the experiment). Reduction of NAD(P)+ was monitored at 340 nm. All the reactions were carried out in a final volume of 50 µl at 25 °C in a 384-microplate reader (Multiskan Ascent, Thermo Electron Corporation). One unit of enzyme activity (U) is defined as the amount of enzyme catalyzing the reduction of 1 µmol NAD(P)+ in 1 min under the specified assay conditions.


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To determine the apparent kinetic constants, enzyme activity was measured at varying concentrations of one substrate at a fixed (unless otherwise stated, saturating) concentration of the second substrate. Data of initial velocity (v) were plotted versus substrate concentration and fitted to the Briggs-Haldane equation: v = Vmax S / (KM + S), where KM is the concentration of substrate (S) producing 50% of the maximal velocity (Vmax).28 Fitting were performed by a nonlinear least-squares algorithm provided by the software Origin 9.0 (OriginLab Corporation). Apparent kinetic constants (and their corresponding standard errors) were calculated by the fitting software using the average of 2 or 3 independent datasets. To better compare the kinetics of the WT and mutant enzymes, we used three different parameters: i) Coenzyme Specificity Ratio (CSR), ii) Relative Catalytic Efficiency (RCE), and iii) Relative Specificity (RS). CSR is the ratio between the catalytic efficiencies for NADP+ and NAD+ for each enzyme (Equation 1). Thus, CSR values higher than 1 are indicative of preference for NADP+. RCE compares the catalytic efficiencies of the mutant enzymes with NADP+ against the WT with NAD+ (Equation 2). RS is the ratio between CSR values of the mutant and WT enzymes (Equation 3). RCE and RS values for the WT enzyme were arbitrarily defined as 1.

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Biochemistry

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Protein thermal shift assays To analyze NAD(P)+ binding to WT and mutant GolDHases, we used the method described by Huynh et al.29 Assays were performed with 0.15 mg mL-1 protein, 4X Sypro Orange (Sigma), 5 mM NAD(P)+, and 20 mM Tris-HCl pH 8.0 in a final volume of 20 μl in 96-well PCR plates (Applied Biosystems). We used two different controls: no protein control (composed of buffer, water, and dye) and ligand only control (which contained ligand, buffer, water, and dye). Plates were sealed with Microseal „B‟ adhesive seals (Bio-Rad) and heated in a StepOnePlus RealTime PCR System (Applied Biosystems) from 25 to 95 °C in increments of 0.2 °C (changes in fluorescence were monitored simultaneously) set for ROX Reference Dye. Homology modeling We used Modeller 9v2 (https://salilab.org/modeller/) to build a 3D-model of peach GolDHase using human GolDHase in complex with NAD+ (PDB ID 1PL8) as template (42.5% identity at the amino acid level between both proteins). The accuracy of the model was checked using the Verify3D server (http://nihserver.mbi.ucla.edu/Verify_3D/). Figure 1 was prepared using UCSF Chimera 1.13.1 (https://www.cgl.ucsf.edu/chimera/).

RESULTS AND DISCUSSION Analysis of cofactor specificity on GolDHases


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We previously reported the kinetic properties of peach GolDHase.18 Herein, we further explore the cofactor specificity and design mutants with altered cofactor specificity based on publicly available structural information.20,30 The WT enzyme and all mutants produced in this work were highly purified in a single step (Figure 2A). As shown in Figure 2B, all recombinant enzymes migrated as tetramers in the gel filtration column. Previous studies suggested that plant GolDHases harbor two Zn2+ ions per subunit, one involved in the catalytic mechanism and a second one important for the structure.31,32 However, to the best of our knowledge, this has not been experimentally demonstrated. Using the Zincon reagent we determined that each subunit contains two Zn2+ ions, thus confirming in silico predictions (Table S2).32,33 These results suggest that all mutants obtained in this work have similar structural properties than the WT.


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Biochemistry

Figure 2. Enzyme purification and quaternary structure determination. (A) Proteins (1 µg) were electrophoresed on a 12% SDS-PAGE. (B) Calibration curve of the Superdex 200 column (open squares) and Kav values for all the enzymes analyzed in this work: grey circle, WT; black triangle, G196A; red triangle, D216A; and blue diamond, D216A/V217R/D218S.

To analyze the cofactor specificity of peach GolDHase, we assayed its activity with either 5 mM NAD+ or NADP+, 200 mM Gol and 2 μM enzyme. As shown in Figure 3A, enzyme activity was 420-fold higher with NAD+ than with NADP+. Previous reports suggested that plant


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GolDHases were not able to use NADP+.17,34,35 Several factors could account for the negative results reported by other authors, such as the low concentrations of NADP+ (1 mM) and enzyme included in the assays, as well as the use of crude extracts instead of pure enzyme preparations.17,34,35 The activity displayed by peach GolDHase with NADP+ (Figure 3A) was high enough to determine the apparent kinetic constants for NADP+ (Tables 1 and S4 and Figure S3). It is important to note that all kinetic parameters reported in this work are apparent values, as they were obtained at fixed concentrations of the second substrate (see above). Gol curves did not reach saturation when using NADP+ as cofactor. Therefore, apparent kinetic constants could not be calculated for this substrate, whereas those calculated for NADP+ were obtained at a fixed (although not saturating) Gol concentration. The kcat was 230-fold higher with NAD+ than with NADP+, while the KM for NAD+ was 8-fold lower than for NADP+ (Table S4). The catalytic efficiency for NAD+ was 1800-fold higher than for NADP+ (Table 1), which clearly shows the preference of peach GolDHase for NAD+. To identify the structural traits that determine cofactor preference in plant GolDHases, we aligned 43 sequences from different species (detailed information is presented in Table S3 and Figure S1). As shown in Figure 3B, site 1 from plant GolDHases display the sequence [V/I/L]X3GAGPIGX6[A/S/V], which matches the consensus sequence described for NAD+binding proteins.13 Analysis of the amino acid frequency shows that the -4 position at site 1 is mostly occupied by Val (95.3%), while the +13 position is mainly occupied by Ala (74.4%). Interestingly, the core of site 1 (GAGPIG) is strictly conserved in all sequences (Figures 3B and S2).


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Biochemistry

Figure 3. Analysis of cofactor specificity of peach GolDHase. (A) GolDHase activity was measured using 200 mM Gol, 5 mM NAD+ or NADP+, and 2 μM enzyme. Assays were performed at pH 9.0 and at 25 °C, in the Gol oxidation direction. (B) WebLogo corresponding to the βαβ subdomain of the Rossmann fold. The full alignment is presented in Figure S1 and sequence information in Table S3. Amino acids were colored as follows: green, basic; grey, hydrophobic; red, acid; blue, polar. Different letters indicate statistically significant differences by t-test (p

Cofactor Specificity Switch on Peach Glucitol Dehydrogenase

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