Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8-+Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8-->Pro and Val-10-*Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.

The cDNAs encoding two different anionic forms of Atlantic cod trypsinogen have been isolated and sequenced. The nucleotide sequences include the 5'-noncoding and 3'-noncoding regions in addition to preproenzymes of 241 amino acids. These consist of hydrophobic signal peptides, activation hexapeptides and trypsins of 222 amino acid residues. The cod trypsins contain all the major structural features common to trypsins such as the catalytic triad His57, Asp102 and Ser195. Furthermore, the obligatory Asp189 and the six disulphide bonds are conserved. Eight amino acid residues are different between the isozymes, leading to a difference of four charges. Both cod trypsins are one-amino-acid-residue shorter than most mammalian trypsins as a result of deletion of proline at position 152, and have a high methionine content. In addition, the cod preproenzyme signal and activation peptides differ markedly from their mammalian analogues. The amino acid identity between the cod and bovine trypsins is approximately 60%.Trypsin, chymotrypsin and elastase belong to the serineprotease family. In addition to an essential serine residue at their active site, they share an identical catalytic mechanism. The mammalian serine proteases are among the most thoroughly studied enzymes, both structurally and functionally.Trypsins from various species are highly similar with respect to their amino acid sequences [l -51. Also, the threedimensional structures available for mammalian trypsins have revealed a strong structural similarity among these enzymes [6, 71. Thus, the trypsins provide a useful model system for studying the relationship between sequence, structure and function.Three anionic trypsin isozymes, termed trypsins I, 11, and 111 with isoelectric points of 6.6, 6.2 and 5.5, respectively, have been isolated from the pyloric caeca of Atlantic cod [8]. All three isozymes showed higher catalytic efficiencies (kca,/ K,) than their bovine counterparts over a range of different temperatures. The catalytic efficiency of the most active form of cod trypsin (trypsin I) was 17-fold higher at 25°C than that of bovine trypsin. Furthermore, the isozymes were less thermostable and acid stable than the bovine analogue [8].Other serine proteases, such as chymotrypsin [9] and elastase [lo] isolated from the Atlantic cod show similar kinetic and temperature characteristics in comparison to their mammalian analogues. Thus, the cod serine proteases have been classified as cold-adapted (pshychrophilic) enzymes.Several other fish trypsins have been characterized with respect to kinetic parameters [ll -141, isoelectric points [13-161 and amino acid composition [13, 15-17] Here we present the cDNA sequences and deduced amino acid sequences of two different trypsin clones isolated from the pyloric caeca of Atlantic cod. One qf the clones appears to be trypsin I previously isolated by Asgeirsson et al. [8], but the other may be either trypsin I1 or trypsin 111. In addition to the trypsinogen-coding region, the nucleotide sequences include the 5'-nontr...

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.

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