Methylmalonic acid

CloneAmp HiFi PCR Premix was from Takara. Restriction enzymes were from New England Biolabs. All primers were synthesized by Elim Biopharm or Sigma-Aldrich. For DNA purification, the GeneJET Plasmid Miniprep Kit and the GeneJET Gel Extraction Kit from Thermo Scientific were used. Stellar Competent Cells were from Takara and One Shot BL21 (DE3) Cells were from Invitrogen. All chemicals for buffer preparations were from Sigma-Aldrich. Isopropyl-β-d-1-thiogalactopyranoside (IPTG), kanamycin sulfate, and carbenicillin were from Gold Biotechnology. LB-Miller Broth for cell cultures was from Fisher Scientific. Ni-NTA affinity resin was from MC Lab. For anion exchange chromatography the HiTrapQ column was from GE Healthcare. For all SDS-PAGE analyses, SDS-PAGE Mini Protean TGX precast gels were from Bio Rad (4–20% and 7.5%). For protein concentration Amicon Ultracentrifugal filters were from Millipore. Coenzyme A (CoA), reduced β-nicotinamide adenine dinucleotide 2′-phosphate (NADPH), sodium propionate, propionic acid, methylmalonic acid, malonic acid, and magenesium chloride hexahydrate were from SigmaAldrich. Adenosine-5′-triphosphate (ATP) was from Teknova. Reducing agent tris(2-carboxyethyl)phosphine (TCEP) was from Thermo Scientific. UVette cuvettes (2 mm ×10 mm path) were from Eppendorf.

Coenzyme A (CoASH), reduced β-nicotinamide adenine dinucleotide 2′-phosphate (NADPH), methylmalonic acid, N-ethylmaleimide, butyryl-CoA, sodium propionate, sodium phosphate, and magnesium chloride hexahydrate were from Sigma-Aldrich. Adenosine-5′-triphosphate (ATP) was from Teknova. Bond-breaker tris(2-carboxyethyl)phosphine (TCEP) was from Thermo Scientific. UVette cuvettes (2 mm × 10 mm path) were from Eppendorf. Methanol, ethyl acetate, and HPLC grade acetonitrile were from Fisher Scientific. Urea was from J. T. Baker. Formic acid was from Fluka. [¹⁴C]-Propionate was from American Radiochemicals. Methylmalonyl-CoA was from Santa Cruz Biotechnology.