d -Alanylation of Lipoteichoic Acid: Role of the d -Alanyl Carrier Protein in Acylation

Abstract

(11,12). Heaton and Neuhaus (12) showed that D-alanyl-Dcp donates its D-alanyl residue to the poly(Gro-P) moiety of membrane-associated LTA. Neither the mechanism nor the topology of this D-alanylation reaction is known. To address this key reaction, several groups have identified the genes in a number of organisms containing the dlt operon (11,21,23). In addition to the genes encoding Dcl (dltA) and Dcp (dltC), dltB and dltD encode a putative transport protein (20) and a protein which facilitates the binding of Dcl and Dcp for ligation with Dalanine and has thioesterase activity for mischarged D-alanyl acyl carrier proteins (ACPs) (5), respectively. A gene encoding an enzyme which catalyzes the transfer of the D-alanine residue from D-alanyl-Dcp to membrane-associated LTA has not been identified.Dcp provides the essential link between the ligase (Dcl) and the incorporation of D-alanine into LTA. This carrier protein is a homologue of those ACPs which function in fatty acid biosynthesis and metabolism (4, 26). However, it was unexpected to find that Dcl will ligate D-alanine to ACPs from Escherichia coli, Vibrio harveyi, Saccharopolyspora erythraea, and Bacillus subtilis (12). Nevertheless, only Dcp participates in the D-alanylation of LTA. These observations suggested that there are at least two determinants for interaction of Dcp with its cognate partners, one of which is recognized by Dcl and one of which is recognized by the membrane acceptor LTA. It is this second determinant which is the focus of structural studies on Dcp (B. F. Volkman, Q. Zhang, D. Debabov, E. Rivera, G. Kresheck, and F. C. Neuhaus, unpublished results), and the biochemical studies to be reported here.For Staphylococcus aureus, it was found that growth in the presence of NaCl resulted in a lower D-alanine ester content in LTA (8). The mechanism by which the ester content was reduced during growth in NaCl is unknown and was not correlated with the reaction catalyzed by DltD (5). Instead, an NaCl-activated, thioesterase-like activity specific for D-alanylDcp which is distinct from that catalyzed by DltD was discovered (5, 20).Our goal here was to establish the mechanism of D-alanine transfer from D-alanyl-Dcp to LTA. To accomplish this goal, the thioesterase-like activity of LTA specific for D-alanyl-Dcp was examined in incubations containing LTA in different microenvironments: (i) purified, (ii) membrane associated, and (iii) membrane associated (salt treated). The results suggested that complex formation between D-alanyl-Dcp and LTA is one of the features resulting either in the transfer of D-alanine from D-alanyl-Dcp to LTA when this amphiphile is membrane associated or in the hydrolysis of D-alanyl-Dcp when the LTA is not membrane associated.

A Pantothenate Kinase from Staphylococcus aureus Refractory to Feedback Regulation by Coenzyme A

“…D-Alanine is bound to the phosphopantetheinyl prosthetic group of Dcp as a thioester (21,22,38), the formation of which is catalyzed by D-alanineDcp ligase (38). Dcp inactivation would block D-alanine incorporation into the cell wall (22,23,38). This biochemical pathway is critical for the functions of the cell wall related to pathogenesis (23), but in the laboratory environment the lack of D-alanine modification is not lethal.…”

“…The hydrophilic backbone of teichoic acids is made up of polymers of glycerol or ribitol joined by phosphate groups, and the extent of esterification with D-alanine determines the net anionic charge and the properties of the cell wall (23). D-Alanine is bound to the phosphopantetheinyl prosthetic group of Dcp as a thioester (21,22,38), the formation of which is catalyzed by D-alanineDcp ligase (38). Dcp inactivation would block D-alanine incorporation into the cell wall (22,23,38).…”

“…E. coli AcpS specifically modifies the ACP involved in fatty acid biosynthesis, although it also accepts substrates such as Dcp and some ACPs of type II polyketide synthases (17,19,20). Dcp is a small carrier protein required for the transfer of D-alanine to lipoteichoic acid in Gram-positive bacteria (21)(22)(23).…”

Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis

“…Its remote homologues include the acyl-coenzyme A synthetases and firefly luciferases 20 . DltA catalyzes the ATP-driven adenylation of the carboxyl group of D-alanine and the transfer of the activated D-alanyl to the thiol group of 4’-phosphopantetheine which is covalently attached to a serine side chain of D-alanyl carrier protein DltC (~80 amino acid residues) 18, 21, 22 . The functional role has not been firmly established for DltB (~400 amino acid residues), an integral membrane protein.…”

Regulation of d-alanylation of lipoteichoic acid in Streptococcus gordonii

“…An increase in the level of D-alanyl ester on LTA has been reported under low external pH conditions (Ellwood & Tempest, 1972;MacArthur & Archibald, 1984), and a decrease in D-alanyl ester contents has been reported when metal cations, such as Mg 2+ , Ca 2+ , Na + and K + , are present in the culture medium (Fischer & Rösel, 1980;Heptinstall et al, 1970;Kiriukhin & Neuhaus, 2001;Koch et al, 1985; Koprivnjak et al, 2006;Reusch & Neuhaus, 1971). An alteration in the enzymic activity of esterification and hydrolyis by these environmental conditions has been suggested as a mechanism for the modulation (Haas et al, 1984;Kiriukhin & Neuhaus, 2001), but transcriptional control of dlt expression has also been reported in three bacteria. In Bacillus subtilis, the dlt operon is transcribed from a s D -dependent promoter, with greatest transcription during exponential growth (Perego et al, 1995 Kuroda et al, 2003;Li et al, 2002;Martínez et al, 2007;Mascher et al, 2004;Suntharalingam et al, 2009).…”