2.3 Enzymatic assays and reaction product analysis

Carboxylate reductase activity of purified CARs against different carboxylic acids and aldehyde reductase activity of purified AKRs were determined spectrophotometrically using an NADPH oxidation-based assay by following the decrease in absorbance at 340 nm. CAR screening was performed in a reaction mixture (0.2 ml) containing HEPES-K (100 mM, pH 7.5), 1 mM NADPH, 2.5 mM ATP, 10 mM MgCl₂, 10 mM substrate (5 mM for decanoic acid), and 2.5 – 5.0 μg of purified CAR (10 min incubation at 30°C). AKR screening was carried out in a reaction mixture containing 50 mM HEPES-K (pH 7.5), 0.5 mM NADPH or NADH, 10 mM butyraldehyde or adipaldehyde, and 2 μg of purified AKR (10 min incubation at 30°C). The pH dependence of reductase activity of purified CARs and AKRs was determined using a mixed buffer system (MEGA buffer) [36]. The kinetic experiments for the determination of K_m and k_cat were performed using a microplate-based assay (0.2 ml) in triplicate in a reaction mixture containing a range of variable substrates (0.025 – 50 mM). Kinetic parameters were determined by non-linear curve fitting from the Lineweaver-Burk plot using GraphPad Prism (version 5.00 for Windows, GraphPad Software, San Diego, CA).

The reaction products of the CAR and AKR catalyzed bioconversions were quantified using a Varian ProStar HPLC system equipped with an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad) and Varian PDA (model 330) and RI (model 350) detectors. Reaction mixtures and culture supernatants were filtered through 10 kDa spin filters (PES membrane, VWR) and eluted using 5 mM H₂SO₄ as the running buffer (0.5 ml/min, 50°C). Concentrations of substrates and products for in vivo and in vitro biotransformations were determined using linear regression analysis of the refractive index peak areas (with subtracted control sample areas). Results are means ± SEM (standard error of mean) from at least three independent determinations.

PolyP kinase reactions with the indicated PPK2 proteins were performed using 5 mM polyP (sodium polyphosphate crystals, +80 mesh, FW ~1286, Aldrich cat # 305553) and 5 mM AMP or 5 mM ADP as substrates. The reaction products (ADP and ATP) were analyzed using reversed phase chromatography on a Varian ProStar HPLC system equipped with a Varian Pursuit C18 column as previously described [37, 38]. Standard solutions of AMP, ADP, and ATP were used to confirm the identity of the observed peaks and products. Formate-dependent reduction of NAD⁺ and NADP⁺ by the D222Q/H224N double mutant FDH from Pseudomonas sp. strain 101 (Uniprot ID {"type":"entrez-protein","attrs":{"text":"P33160","term_id":"4033692","term_text":"P33160"}}P33160) was followed at 340 nm [39]. Reaction mixtures contained 100 mM Tris-HCl (pH 8.0), 50 mM formate, and 1 μg of purified mutant FDH.

Liquid chromatography-mass spectrometry (LC-MS) analysis of CAR reaction products (1,4-BDO, 1,6-HDO, and 6-HHA) was performed using a Dionex Ultimate 3000 UHPLC system and a Q-Exactive mass spectrometer equipped with a HESI source (all from Thermo Scientific) and controlled by Thermo XCalibur 2.2 software. LC separation was conducted on a Hypersil Gold C18 column (50 mm × 2.1 mm, 1.9 μ particle size, Thermo Scientific) equipped with a guard column. Solvent A was 0.1% formic acid (in water), solvent B was 0.1% formic acid in methanol (flow rate 0.2 ml/min). Autosampler temperature was maintained at 8°C, and injection volume was 10 μl. The gradient was 0 – 2.5 min: 100% A; 2.5 – 4.5 min: 15% A, 85% B; 4.5 – 8.0 min: 15% A, 85% B; 8.0 – 8.5 min: 100% A; 8.5 – 12 min: 100% A. Data collection was done in positive ionization mode with a scan range m/z 100–300, resolution 70000 at 1 Hz, AGC target of 3e6 and a maximum injection time of 200 ms. Standard solutions of 1,4-BDO (m/z 91.0769), 1,6-HDO (m/z 119.1062), and 6-HHA (m/z 133.0864) were used for validation of retention time and m/z.