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

Escherichia coli acyl carrier protein (ACP) contains a single tyrosine resi due at position 71. The combined o-nitration of apo-ACP Y71 by tetranitrome thane 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 report ed. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated an d acylated in vitro with an overall similar to 30% yield of purified stearo yl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-stat e 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 w ell for studying binding interactions with the Delta (9)-desaturase and sug gest similar possibilities for other ACP-dependent enzymes. The efficient i n vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with hol o-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported. (C) 2000 Academic Press.