Catalytic properties of a newly discovered acyltransferase that synthesizes N-acylphosphatidylethanolamine in cottonseed (Gossypium hirsutum L.) microsomes

We recently demonstrated that cotyledons of cotton (Gossypium hirsutum L.) seedlings synthesize N-acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), during postgerminative growth (K.D. Chapman and T.S. Moore [1993] Arch Biochem Biophys 301: 21-33). Here, we report the discovery of an acyltransferase enzyme, fatty acid: diacylphosphatidylethanolamine N-acyltransferase (designated NAPE synthase), that synthesizes NAPE from PE and free fatty acids (FFA) in cottonseed microsomes. [14C]NAPE was synthesized from [14C]palmitic acid and endogenous PE in a time-, pH-, temperature-, and protein concentration-dependent manner. [14C]palmitic acid was incorporated exclusively into the N-acyl position of NAPE. [14C]palmitoyl coenzyme A (CoA) and [14C]-dipalmitoyl phosphatidylcholine (PC) were poor acyl donors for the synthesis of NAPE (i.e. 200- and 3000-fold lower incorporation efficiency than palmitic acid, respectively). Synthesis of NAPE f rom palmitoyl-CoA and dipalmitoyl-PC was observed only after the release of FFA in microsomes. We observed a temperature optimum of 45 degrees C and a pH optimum of 8.0 for the synthesis of [14C]NAPE from [14C]palmitic acid (or from [14C]PE). NAPE synthase activity showed no apparent divalent cation requirement. Notably, activity was stimulated by HPO4(2-), HCO3(-), SO4(2-), and NADPH, whereas activity was inhibited by Ca2+, Mn2+, Co2+, Cd2+, ATP, ADP, flavin adenine dinucleotide, and flavin mononucleotide. Other nucleotide triphosphates (GTP and CTP) and pyridine dinucleotides (NAD, NADH, and NADP) did not appreciably affect NAPE synthase activity. Initial velocity measurements of NAPE synthase activity at increasing concentrations of palmitic acid showed non-Michaelis-Menten, biphasic kinetics. A high-affinity site (S(0.5) = 7.2 micromolars, Vmax = 18.8 nmol h-1 mg-1 of protein) and a low-affinity site (S(0.5) = 32.0 micromolar, Vmax = 44.9 nmol h-1 mg-1 of protein) were identified