Chemical and posttranslational modification of Escherichia coli acyl carrier protein for preparation of dansyl-acyl carrier proteins'

摘要

Escherichia coli acyl carrier protein (ACP) contains a single tyrosine residue at position 71. The combined o-nitration of apo-ACP Y71 by tetranitromethane and reduction to 3-aminotyrosyl-apo-ACP were performed to introduce a specific site for attachment of a dansyl fluorescent label, Conditions for purification and characterization of dansylaminotyrosyl-apo-ACP are reported. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated and acylated in vitro with an overall similar to 30% yield of purified stearoyl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-state kinetic parameters k(cat) = 22 min(-1) and K-M = 2.7 muM were determined for reaction of stearoyl-dansylaminotyrosyl-ACP with stearoyl-ACP Delta (9)-desaturase. These results show that dansylaminotyrosyl-ACP will function well for studying binding interactions with the Delta (9)-desaturase and suggest similar possibilities for other ACP-dependent enzymes. The efficient in vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with holo-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported. (C) 2000 Academic Press. [References: 32]