Malate dehydrogenase

For TER activity measurement, the reaction mixture consisted of 50 mM potassium phosphate pH 7.5, 0.1 mg/ml bovine serum albumin (BSA), 0.1% GDN, 2 µM FAD, 5 mM MnCl₂, 5 mM MgCl₂, 200 µM crotonyl-CoA or 2-trans-dodecenoyl-CoA, 20 nM SC I + III₂ + IV or 50 µg/ml E. gracilis isolated mitochondrial membranes or 50 µg/ml Homo sapiens 2-enoyl thioester reductase 1 (ETR1) (Solarbio Life Science). The reaction was started by the addition of 250 µM NAD(P)H, mixed in the spectrophotometer for 10 s and the 340 nm absorbance was recorded every 6 s for above 10 min. The TER activity was calculated as NAD(P)H oxidation rate based on the slope of the initial linear phase between 0–300 s in the 340 nm kinetic curve. For PEPCK activity measurement, the reaction mixture contained 30 mM Tris pH 7.4, 100 mM NaCl, 100 mM NaHCO₃, 5 mM MnCl₂, 5 mM MgCl₂, 500 µM NADH, 10 mM ADP or GDP, 1.2 U malate dehydrogenase (Sigma-Aldrich), 20 nM Eg-SC I + III₂ + IV or 50 µg/ml E. gracilis isolated mitochondrial membranes or 50 µg/ml Zea mays PEPCK (Shanghai Yuanye Bio-Technology). The reaction was started with the addition of 10 mM PEP, mixed in the spectrophotometer for 10 s and the 340 nm absorbance was recorded every 6 s for above 10 min. The PEPCK activity was calculated as NADH oxidation rate based on the slope of the initial linear phase between 0–300 s in the 340 nm kinetic curve.

The decarboxylase activity of wild-type and mutant MdcD–MdcE complexes toward malonyl-CoA was determined using a coupled-enzyme assay, converting acetyl-CoA production to NAD⁺ reduction^{46 (link)}. The reaction contained 2.5 μM MdcD–MdcE, 6 mM l-malate, 0.5 mM NAD⁺, 1 U malate dehydrogenase (Sigma), 0.2 U citrate synthase (Sigma), 20 mM Tris (pH 8.0), and various concentrations of malonly-CoA. The production of NADH was monitored by absorbance at 340 nm.

ClpB was amplified by PCR and inserted into pDS56 and verified by sequencing. Mutant derivatives of clpB were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing. ClpB was purified after overproduction from E. coli ΔclpB::kan cells. ClpB wild-type and mutant variants were purified using Ni-IDA (Macherey-Nagel) and size exclusion chromatography (Superdex S200, Amersham) following standard protocols. Purifications of DnaK, DnaJ, GrpE, Luciferase and Casein-YFP were performed as described previously (Haslberger et al., 2008 (link), Oguchi et al., 2012 (link), Seyffer et al., 2012 (link)). Pyruvate kinase of rabbit muscle and Malate Dehydrogenase of pig heart muscle were purchased from Sigma. Protein concentrations were determined with the Bio-Rad Bradford assay.

Pck activity was determined as previously described [9 (link)]. To monitor the rate of the anaplerotic reaction (OAA formation), the Pck-catalyzed reaction was coupled with the subsequent reaction catalyzed by malate dehydrogenase from Thermus flavus (Sigma-Aldrich). The standard reaction mixture contained 100 mM HEPES-NaOH, pH 7.2, 100 mM KHCO₃, 37 mM DTT, 2 mM PEP, 1 mM GDP, 2 mM MgCl₂, 0.1 mM MnCl₂, 2 U/ml MDH, and 0.25 mM NADH. Each reaction was started by addition of the essential divalent cations (Mg²⁺ and Mn²⁺). To monitor the gluconeogenic reaction (PEP formation), the Pck-catalyzed reaction was coupled with reactions catalyzed by pyruvate kinase (PK, Roche) and lactate dehydrogenase (LDH, Roche). The typical reaction mixture was composed of 100 mM HEPES-NaOH, pH 7.2, 0.3 mM OAA, 0.2 mM GTP, 2 mM MgCl₂, 0.2 mM MnCl₂, 10 mM DTT, 10 U/ml LDH, 3 U/ml PK, and 0.2 mM NADH. The reaction progress for both reactions was followed by monitoring the decrease in absorbance at 340 nm due to NADH oxidation to NAD⁺. The kinetic data were fitted using a nonlinear least squares regression analysis (SigmaPlot 11.0).