Lactococcus lactis strain NIZO 221 86 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity. Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.Lantibiotics are ribosomally synthesized antimicrobial polypeptides containing the unusual amino acids lanthionine and fl-methyllanthionine [I]. Genetic analysis has shown that the lantibiotics are produced as precursor molecules consisting of an uncommon leader region and a structural region that is post-translationally processed [1 -51. Nisin is a 34-aminoacid lantibiotic produced by Lactococcus lactis that has wide applications as a food preservative 161. It also contains dehydroalanine and dehydrobutyrine (P-methyldehydroalanine) [7]. An extensive characterization of nisin by onedimensional and two-dimensional NMR analysis has recently been described [8, 91. Long range contacts were observed in the NOE spectra of nisin, indicating the presence of a preferred conformation [8]. In order to obtain further insight into the structure/function relationship of nisin, we purified and characterized an antimicrobial peptide produced by L. luctis strain NIZO 22186 that showed high similarity to nisin. Our detailed analysis on the biochemical and genetic level shows that this compound is a natural nisin variant, designated nisin Z, that differs from nisin in a single amino acid. MATERIALS AND METHODS Cultivation and purification L . luctis, subspecies luctis, strains NIZO R5 [lo] and NIZO22186 (NIZO collection) were grown for 16 h at 30°C in a medium containing 1% sucrose, 1% peptone, 1% yeast extract, 0.2% NaCI, 0.002% MgS04 . 7 H 2 0 and 1 YO KH2P04, pH 6.8 (L. de Vuyst, personal communication). After addition of 0.5 M (NH4)2S04, the cell-free supernatant (5 1) was run on a Fractogel TSK butyl650-S (Merck) column (5 x 20 cm), Correspondence to W. M. de Vos, Netherlands Institute for DairyAbbreviations. RP, reversed phase; Ile, isoleucine. Note. The novel nucleotide sequence data published here has been deposited with the EMBL sequence data banks and is available under accession number X61144.The novel amino acid sequence data published here has been deposited with the EMBL sequence data bank.Research (NIZO), P.O. Box 20, NL-6710 BA Ede, The Netherlands which was washed with 1.51 0.5M (NH4)2S04 and subsequently with deionized water until A220 < 0.5 was achieved. The antimicrobial substance was then eluted using 10mM HC1 and concentrated by ultrafiltration (Filtron, Novacell, Omega, molecular mass cut off 1 kDa). Final purification and analy...

Antithrombin is a member of the serine proteinase inhibitor (serpin) family which contain a flexible reactive site loop that interacts with, and is cleaved by the target proteinase. In cleaved and latent serpins, the reactive site loop is inserted into a large central beta-sheet in the same molecule, whereas in ovalbumin, a nonfunctional serpin, the reactive site loop is completely exposed and in an alpha-helical conformation. However, in neither conformation can the reactive site loop bind to target proteinases. Here we report the structure of an intact and cleaved human antithrombin complex. The intact reactive site loop is in a novel conformation that seems well suited for interaction with proteinases such as thrombin and blood coagulation factor Xa.

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The major components of mammalian lenses are tissue-specific, soluble proteins, the alpha-, beta- and gamma-crystallins. The lenses of other vertebrate classes often contain other major proteins, notably delta-crystallin in birds and reptiles. A fourth distinct type, described as epsilon-crystallin, is prominent in many bird and crocodile lenses. Here we show that epsilon-crystallin is an active glycolytic enzyme, lactate dehydrogenase (LDH) (EC 1.1.1.27) and that duck epsilon-crystallin appears to be identical to duck LDH-B4. LDH is a normal metabolic component in other lenses, but in duck is present in amounts far exceeding the requirements of any likely catalytic role. It appears that an active enzyme has been recruited, unchanged, to an extra role as a structural protein in the lens without gene duplication and sequence divergence. This surprising discovery raises the possibility that other crystallins may similarly be enzymes expressed at high levels in lens as structural proteins.

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