D-alanylation of lipoteichoic acid: Role of the D-alanyl carrier protein in acylation

The D-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-po sitive organisms requires the D-alanine-D-alanyl carrier protein ligase (AM P) (Dcl) and the D-alanyl carrier protein (Dcp). The dlt operon encoding th ese proteins (dltA and dltC) also includes dltB and dltD, dltB encodes a pu tative transport system, while dltD encodes a protein which facilitates the binding of Dcp and Dcl for ligation with D-alanine and has thioesterase ac tivity for mischarged D-alanyl-acyl carrier proteins (ACPs), In previous re sults it was shown that D-alanyl-Dcp donates its ester residue to membrane- associated LTA (M, P, Heaton and F, C, Neuhaus, J, Bacteriol. 176: 681-690, 1994), However, all efforts to identify an enzyme which catalyzes this D-a lanylation process were unsuccessful. It was discovered that incubation of D-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolys is of this D-alanyl thioester, D-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated D-alanyl LTA, a time and concentration-dependent formation of D-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of D-alanyl-Dcp a nd stimulated the hydrolysis of D-alanyl-Dcp, Since these reactions are spe cific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the D-al anylation process. The reversibility of this process provides a mechanism f or the transacylation of the D-alanyl eater residues between LTA and wall t eichoic acid.