(ω–2) Hydroxylation of Fatty Acids by a Soluble System from Bacillus megaterium

Abstract

Abstract A cell-free hydroxylating system which acts on long chain fatty acids has been isolated from Bacillus megaterium. When palmitic acid is used as a substrate, the major product is 14-hydroxypalmitate (about 50% of the total) while 15-hydroxypalmitate (30%) and 13-hydroxypalmitate (20%) are also produced. No 16-hydroxypalmitate is formed in this system. The effect of substrate chain length on hydroxylation activity was tested with various saturated fatty acids. The relative activity of the hydroxylase with the substrates tested was as follows: C15 (1.00), C16 (0.89), C14 (0.72), C17 (0.34), C18 (0.17), and C12 (0.08). When the distributions of isomeric hydroxy acids formed from C18 and C14 were determined, it was found that the major product from C18 was the (ω - 2) derivative with the (ω - 1) and (ω - 3) components being present in about the same ratio as for palmitate. Myristic acid, however, gave approximately equal amounts of the three isomers, with the (ω - 1) derivative being slightly favored. In neither case was the ω-hydroxy acid detected among the products. Several monounsaturated fatty acids were also tested and were found to be more active as substrates than their saturated analogs of the same chain length. The hydroxylation system is soluble and, in crude form, appears to be quite stable and active. All of the hydroxylase activity remains in the supernatant when a cell-free preparation from B. megaterium is centrifuged at 100,000 x g for 2 hours. Free palmitic acid is a much better substrate than the CoA derivative. Both NADPH and O2 are required for hydroxylase activity but no other non-protein cofactor seems to be involved. The hydroxylation activity is stable to dialysis but decreases to about 20 to 25% of the original level when the 100,000 x g supernatant is passed through Sephadex G-25, presumably due to the loss of a small protein component. The activity can be restored completely by the addition of bacterial ferredoxin to the gel-filtered preparation, but the preparation is no longer stable to freezing and thawing or further chromatographic procedures. The endogenous component for which ferredoxin substitutes is not removed by (NH4)2SO4 fractionation of the 100,000 x g supernatant protein followed by Sephadex G-200 chromatography, and a 10- to 11-fold purification of the hydroxylase was achieved in this manner. However, the hydroxylating activity of this preparation was not stimulated by added ferredoxin and further chromatographic procedures designed to remove the endogenous ferredoxin-like component always resulted in irreversible loss of activity.