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Circulatory lipid transport in animals is mediated to a substantial extent by members of the large lipid transfer (LLT) protein (LLTP) superfamily. These proteins, including apolipoprotein B (apoB), bind lipids and constitute the structural basis for the assembly of lipoproteins. The current analyses of sequence data indicate that LLTPs are unique to animals and that these lipid binding proteins evolved in the earliest multicellular animals. In addition, two novel LLTPs were recognized in insects. Structural and phylogenetic analyses reveal three major families of LLTPs: the apoB-like LLTPs, the vitellogenin-like LLTPs, and the microsomal triglyceride transfer protein (MTP)-like LLTPs, or MTPs. The latter are ubiquitous, whereas the two other families are distributed differentially between animal groups. Besides similarities, remarkable variations are also found among LLTPs in their major lipid-binding sites (i.e., the LLT module as well as the predicted clusters of amphipathic secondary structure): variations such as protein modification and number, size, or occurrence of the clusters. Strikingly, comparative research has also highlighted a multitude of functions for LLTPs in addition to circulatory lipid transport. The integration of LLTP structure, function, and evolution reveals multiple adaptations, which have come about in part upon neofunctionalization of duplicated genes. Moreover, the change, exchange, and expansion of functions illustrate the opportune application of lipid-binding proteins in nature.Accordingly, comparative research exposes the structural and functional adaptations in animal lipid carriers and brings up novel possibilities for the manipulation of lipid transport.

The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.

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a-Amylases belong to the alp-barrel protein family in which the active site is created by residues located at the C-terminus of the p strands and in the helix-connecting loops extending from these ends. In the a-amylase family, a small separate domain B protrudes at the C-terminus of the third p strand of the @/a),-barrel framework. The 80% identical barley a-amylase isozymes 1 and 2 (AMYl and AMY2, respectively) differ in substrate affinity and turnover rate, CaC1, stimulation of activity, sensitivity to the endogenous 21-kDa a-amylaselsubtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1 -AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMYl-(l-9O)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161 -residue N-terminal sequences, respectively, of AMY 1 and complementary C-terminal regions of AMY2. AMYl-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to a-amylaselsubtilisin inhibitor and its low affinity for the substrates p-nitrophenyl a-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-( 1-161)-AMY2-(161-403) and AMYl, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaC1,. Domain B thus determines several functional and stability properties that distinguish the barley a-amylase isozymes.a-Amylases hydrolyse internal a-l,4-glucosidic linkages of starch and related dextrins and are widely occurring in microorganisms, higher plants and animals. In cereals, aamylases represent a major starch-degrading activity important in seed germination (MacGregor, 1987). a-Amylases and related amylolytic enzymes are @/a),-barrel proteins (Matsuura et al