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

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