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J Bacteriol. 1992 July; 174(14): 4707-4717

research-article

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme.

M P Heaton and F C Neuhaus

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208.

ABSTRACT

The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.

J Bacteriol. 1992 July; 174(14): 4707-4717




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