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In 2013, Tominaga and co–workers crystallized carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis after in vitro incubation of the protein with potassium cyanide and ATP [27 ].
Enzyme assays were performed in a 100 μL volume containing 50 mM Bicine, pH 8.5, 25 mM potassium acetate, 2 mM MgCl 2 , 0.1% (w/v) bovine serum albumin (BSA), 0.375 mM NADH (Sigma, N8129), 0.7 mM phosphoenolpyruvate tricyclohexylammonium salt (Sigma, P7252), 6 U/ml pyruvate kinase, and 30 U/ml lactate dehydrogenase (both from Sigma, P0294) at 37°C. 10 mM maltotriose (Sigma, M8378) were used as an acceptor.