ROLE OF THE D-ALANYL CARRIER PROTEIN IN THE BIOSYNTHESIS OF D-ALANYL-LIPOTEICHOIC ACID

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) is a widespread macroamphiph ile which plays a vital role in the growth and development of gram-pos itive organisms. The biosynthesis of this polymer requires the enzymic activation of D-alanine for its transfer to the membrane-associated L TA (mLTA). A small, heat-stable, and acidic protein that is required f or this transfer was purified to greater than 98% homogeneity from Lac tobacillus casei ATCC 7469. This protein, previously named the D-alani ne-membrane acceptor ligase (V.M. Reusch, Jr and F. C. Neuhaus, J. Bio l. Chem. 246:6136-6143, 1971), functions as the D-alanyl carrier prote in (Dcp). The amino acid composition, beta-alanine content, and N-term inal sequence of this protein are similar to those of the acyl carrier proteins (ACPs) of fatty acid biosynthesis. The isolation of Dcp and its derivative, D-alanyl similar to Dcp, has allowed the characterizat ion of two novel reactions in the pathway for D-alanyl-mLTA biosynthes is: (i) the ligation of Dcp,vith D-alanine and (ii) the transfer of D- alanine from D-alanyl-Dcp to a membrane acceptor. It has not been esta blished whether the membrane acceptor is mLTA or another intermediate in the pathway for D-alanyl-mLTA biosynthesis. Since the D-alanine-act ivating enzyme (EC 6.1.1.13) catalyzes the ligation reaction, this enz yme functions as the D-alanine-Dcp ligase (Dcl). Dcl also ligated the ACPs from Escherichia coli, Vibrio harveyi, and Saccharopolyspora eryt hraea with D-alanine. In contrast to the relaxed specificity of Dcl in the ligation reaction, the transfer of D-alanine to the membrane acce ptor was highly specific for Dcp and did not occur with other ACPs. Th is transfer was observed by using only D-[C-14]alanyl similar to Dcp a nd purified L. casei membranes. Thus, D-alanyl-Dcp is an essential int ermediate in the transfer of D-alanine from Dcl to the membrane accept or. The formation of D-alanine esters of mLTA provides a mechanism for modulating the net anionic charge in the cell wall.