HIGH-MOLECULAR-WEIGHT PROTEIN-2 OF YERSINIA-ENTEROCOLITICA IS HOMOLOGOUS TO ANGR OF VIBRIO-ANGUILLARUM AND BELONGS TO A FAMILY OF PROTEINS INVOLVED IN NONRIBOSOMAL PEPTIDE-SYNTHESIS

The iron-regulated irp2 gene is specific for the highly pathogenic Yer sinia species and encodes high-molecular-weight protein 2 (HMWP2). Des pite the established correlation between the presence of HMWP2 and vir ulence, the role of this protein is still unknown. To gain insight int o the function of HMWP2, the entire coding sequence and the promoter o f irp2 were sequenced. Two putative -35 and -10 promoter sequences wer e identified upstream of a large open reading frame, and two potential Fur-binding sites were found overlapping the second -35 box. The larg e open reading frame is composed of 6,126 nucleotides and may encode a protein of 2,035 amino acids (ca. 228 kDa) with a pl of 5.81. A signa l sequence was not present at the N terminus of the protein. Despite t he existence of 30 cysteine residues, carboxymethylation prevented the formation of most if not all disulfide bonds that otherwise occurred when the cells were sonicated. The protein was composed of three main domains: a central region of ca. 850 residues, bordered on each side b y a repeat of 550 residues. A high degree of identity (44.5%) was foun d between HMWP2 and the protein AngR of Vibrio anguillarum. The centra l part of HMWP2 (after removal of a loop of 337 residues) also display ed significant homology with proteins belonging to the superfamily of adenylate-forming enzymes and, like them, possessed a putative ATP-bin ding motif that is also present in AngR. In addition, HMWP2 shared wit h the group of antibiotic and enterochelin synthetases a potential ami no acid-binding site. Six consensus sequences defining the superfamily and four defining the family of synthetases were derived from the mul tiple alignment of the 30 sequences of proteins or repeated domains. A phylogenetic tree that was constructed showed that HMWP2 and AngR are in a family composed of Lys2, EntF, and the tyrocidine, gramicidin, a nd beta-lactam synthetases. This finding suggests that HMWP2 may parti cipate in the nonribosomal synthesis of small biologically active pept ides.