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In gram-negative bacteria, the extracellular proteins have two lipid bilayers to cross, in contrast to the proteins targeted to the periplasm or the outer membrane. Depending upon the secretion route and the genes involved, two major secretion pathways can be identified. Without traversing the periplasm, oc-hemolysin of Escherichia coli is secreted through a hemolysin translocator composed of HlyB, HlyD (24), and ToiC (48). Two genes, prtD and prtE, required for the secretion of an extracellular protease by Erwinia chrysanthemi were found to be homologous to hlyB and hlyD, respectively (27). In the second category, extracellular proteins, for example, the pullulanase of Klebsiella oxytoca, are secreted via the periplasm (34). A closely linked operon includes at least 12 pul genes encoding proteins required for the secretion of pullulanase in addition to the sec genes involved in the translocation of proteins across the inner membrane (34, 35). Homologs of the pul genes are also required for the secretion of extracellular proteins by E. chrysanthemi (22), Pseudomonas aeruginosa (15), Xanthomonas campestns pv. campestris (12), and Yersinia enterocolitica (29). Mutations in these genes can result in the accumulation of extracellular proteins in the periplasm (1,7,11,16,29,34,45,49

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of L-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn 2؉ ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg 2؉ , Mn 2؉ , Co 2؉ , or Zn 2؉ , but Ca 2؉ has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg 2؉ ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca 2؉ .The histidine biosynthetic pathway serves as a model system for better understanding of the fundamental metabolic, physiological, and genetic processes in bacteria (1). This pathway is identical in both Escherichia coli and Salmonella typhimurium and has been thoroughly characterized (1, 2). The sixth and eighth steps of histidine biosynthesis are catalyzed by imidazole glycerol phosphate dehydratase (IGPD, 3 EC 4.2.1.19) and histidinol phosphate phosphatase (HPase, EC.3.1.3.15) respectively (1) (Scheme 1). In protobacteria, including E. coli and S. typhimurium, the IGPD and HPase activities are encoded by a single gene (3-5), whereas in archaea, eukarya, and most bacteria they are encoded by two separate genes (3). The bifunctional HisB enzyme has been proposed to be the result of a fusion of two independent cistrons that occurred recently in evolution (3).Biochemical and genetic studies of the HisB enzyme together suggest that both of its enzymatic activities are independent of one another and reside in separate domains (6 -8). The HPase activity is found within the N-terminal domain (residues 1-167, HisB-N), whereas the C-terminal domain (residues 168 -356) exhibits the IGPD activity. The phosphatase activity requires the presence of metal ions such as Mg 2ϩ , Mn 2ϩ , Co 2ϩ , or Zn 2ϩ but is inhibited by calcium (9). Based on the presence of four invariant aspartic acid residues, HisB has been classified as a member of the haloacid dehalogenase-like hydrolase (HAD) family within the "DDDD" superfamily of aspartyl-phosphate utilizing phosphohydrolases/phosphotransferases (10, 11). The phosphoryl transfer catalyzed by phosphotransferases and phosphatases from the HAD family occurs via a phosphoa...

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The last ORF of an xps gene cluster, designated xpsD, is required for the secretion of extracellular enzymes across the outer membrane in Xanthomonas campestris pv. campestris. It could encode a protein of 759 amino acid residues. A consensus N-terminal lipoprotein signal peptide was revealed from its deduced amino acid sequence. A [3H]palmitate labelling experiment indicated that XpsD was fatty-acylated. Differential extraction with Triton X-100 disclosed that XpsD was fractionated with the outer membrane. Sucrose gradient sedimentation analysis of total membranes also indicated that XpsD was mainly located in the outer membrane. At least part of XpsD is exposed to the cell surface as suggested by trypsin experiment results. Intact cells pretreated with antibody against XpsD could indirectly be labelled with fluorescent agent. When the N-terminal lipoprotein signal peptide was replaced with a nonlipoprotein signal peptide cleavable by signal peptidase I, non-fatty-acylated XpsD was synthesized. Its subcellular location was indistinguishable from that of the fatty-acylated XpsD. Complementation of an xpsD::Tn5 mutant of X. campestris pv. campestris indicated that this non-fatty-acylated XpsD remains functional in extracellular protein secretion. A stable, C-terminal truncated protein, XpsDd414-759, was synthesized from a mutated xpsD gene. Although it stayed associated with the outer membrane and exposed to the cell surface, it no longer could complement the xpsD::Tn5 mutant of X. campestris pv. campestris.

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Two genes encoding ␤-galactosidase isoenzymes, ␤-galI and ␤-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6-and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. ␤-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5 flanking region of ␤-galI.

&-Amylase (1,4-a-D-ghcan glucanohydrolase, EC 3.2.1.1) of apparent molecular mass 45 kDa was secreted by Xanthomonas campestris pv. campestris grown in medium containing starch or maltose. We isolated its structural gene from a recombinant A library and located it on a 2.7 kb DNA fragment. Nucleotide sequencing of the fragment revealed a potential ORF encoding a protein of 475 amino acid residues, including a potential signal sequence of 35 amino acids. The signal processing site was confirmed by N-terminal amino acid sequence analysis of the exported a-amylase. The deduced amino acid sequence of the mature protein is very similar to that of the a-amylase of Aeromunas hydrophifa It also contains all four amino acid sequences highly conserved in the aamylases from a wide range of organisms. Expression of the amy gene in Escherichia cufi was poor from its own promoter, but was enhanced by the upstream promoter on the vector. The a-amylase synthesized in E. cofi was located in the periplasm.