The active site of the enzyme is tentatively identified as a pocket at the carboxy-terminal end of the eight-stranded beta-barrel. Lys165 lies within this pocket and is probably the reactive residue which forms a Schiff base intermediate with the substrate. The sequence of N-acetylneuraminate lyase has similarities to those of dihydrodipicolinate synthase and MosA (an enzyme implicated in rhizopine synthesis) suggesting that these last two enzymes share a similar structure to N-acetylneuraminate lyase.
The amino acid sequence of a methionine-rich 2s seed protein from sunflower (Heliunthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2s proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2s protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2s proteins have diverged as a subclass of the 2s superfamily of proteins which contain only 2-3% methionine. While the related 2s proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.The seed storage proteins of most dicotyledonous plants contain two major protein classes, globulins and albumins, which are distinguished on the basis of solubility [l]. These proteins are expressed during seed development as precursors which undergo co-and post-translational modification prior to deposition in protein bodies [2]. The globulins, which are soluble in high-salt buffers and have sedimentation coefficients of 7s and 11S, are characterized by high levels of arginine, glutamine and asparagine, which provide a source of nitrogen for the developing seedling [2].The albumins are a more diverse group of proteins, usually soluble in water, with sedimentation coefficients of approximately 2s and some are rich in cysteine. While some 2s proteins appear to have only a storage function, some others such as lectins and protease and a-amylase inhibitors have biological activity. As a consequence of this these proteins are nutritionally undesirable. The sequences of a number of 2s seed storage proteins from different plants have shown that some of these proteins share sequence similarities and they have been grouped into a 2s superfamily of related sulfur- (EC 3.4.21.19).Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X56686. The novel amino acid sequence data have been deposited with the EMBL sequence data bank. rich seed proteins [3]. The 2s proteins from castor bean [4], rapeseed [5], mustard seed [6] Brazi...
An in vitro system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized in vitro from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The in vitro assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein.
A DNA fragment encoding an extracellular basic protease (PI M 9.5) from Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative agent of ovine footrot, has been cloned and expressed in Escherichiu coli and sequenced. E. coli harbouring a plasmid with a 3-kb DNA fragment containing the D. nodosus basic-protease gene exhibited proteolytic activity when tested on skim-milk plates. The sequence of the native basic protease isolated from D. nodosus was also determined by direct amino acid sequencing. Comparison of the deduced sequence of the primary translation product (603 residues) and that of the native protease (344 residues) indicates that the protease is synthesized as a precursor molecule, containing a signal peptide (21 residues), a 11 1 amino acid pro-peptide and a 127 residue C-terminal extension which is subsequently processed to the mature active form.Comparison of the D. nodosus basic protease sequence with that of other serine proteases showed that it is related to the subtilisin family of proteases with strong conservation of sequence identity around the catalytic site residues. A remarkable similarity in structure was found to the serine protease of Xunthomonus cumpestris, a plant pathogen, with respect to the length of the precursor segments, conservation of disulfide bridges and approximately 50% sequence identity of the mature proteases. Electrophoretic analyses on non-denaturing gels at pH 8.8 of culture supernatants of virulent and benign D. nodosus strains show the presence of four or five proteases with PI in the range 4. 9 -6.0 [18, 191, and SDSjPAGE analyses indicate apparent MI of 40000 and 42000 [20]. The partial purification and properties of these proteases has been described previously [8, 201. Recently, we isolated and purified a serine protease from the culture supernatant of D. nodosus strain 198 which had not been found in the earlier studies. This protease is a basic protein with a PI of ~9 . 5 and a MI ~3 7 0 0 0 and is referred to as the D. nodosus basic protease. It has been isolated from culture supernatants of several different serogroups of virulent D. nodosus strains (234, 265 and 332), but is not present in significant amounts in culture supernatants of benign D. nodosus strains. ELISA and agar immunodiffusion have demonstrated that this basic protease is serologically distinct from the proteases with the PI of 4.9 -6.0 described previously [8, 19, 201. Dichelobacter nodosus
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