Enzyme assay

The physiological, forward reaction of P5C reductase was measured at 30°C following the oxidation of NAD(P)H at 340 nm. Under standard conditions, the assay mixture contained 20 mM Tris-HCl buffer, pH 7.0, 0.5 mM NADH or NADPH, and 1 mM l-P5C in a final volume of 0.2 mL. Proteins were column buffer-exchanged just before the analysis to remove Hepes buffer, which was replaced with 10 mM Tris-HCl buffer, pH 7.0. dl-P5C was synthesized by the periodate oxidation of δ-allo-hydroxylysine (Sigma H0377) and purified by cation-exchange chromatography according to Williams and Frank (1975). A limiting amount of enzyme (about 0.1, 2, 0.4, and 10 μg of the purified ProH, ProI, ProG, and ComER, respectively) was added to the pre-warmed mixture, and the decrease in absorbance at 340 nm was recorded at 30-s intervals and up to 25 min through an optical path of 0.5 cm. Unspecific oxidation of pyridine dinucleotides, measured in parallel blanks in which P5C had been omitted, was subtracted, and activity was calculated from the initial linear rate on the assumption of a molar extinction coefficient for NAD(P)H of 6,220 M⁻¹ cm⁻¹. Linear regression analysis was computed with Prism 6 (version 6.03, GraphPad Software, Inc., USA). Protein content was determined by the Coomassie Blue method (Bradford, 1976), using bovine serum albumin as the standard. For the purified proteins, direct absorbance at 280 nm was used instead, and the concentration was calculated by a deduced (http://web.expasy.org/protparam/) molar extinction coefficient of 9,065, 10,555, 14,565, and 10,805 M⁻¹ cm⁻¹ for ProH, ProI, ProG, and ComER, respectively.