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 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 approximately 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 microM 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.
Haas
J A
JA
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Frederick
M A
MA
Fox
B G
BG
D000213:Acyl Carrier Protein / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000215:Acylation; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003619:Dansyl Compounds / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008958:Models, Molecular; D010204:Pantetheine / Q000031:analogs & derivatives; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013774:Tetranitromethane / Q000378:metabolism
D000213:Acyl Carrier Protein; D001059:Apoproteins; D001426:Bacterial Proteins; D003619:Dansyl Compounds; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C040157:acpP protein, E coli; D010204:Pantetheine; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase; D054889:Fatty Acid Synthase, Type II; D013774:Tetranitromethane