This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Proteins of the transferrin family, which contains serum transferrin and lactoferrin, control iron levels in higher animals through their very tight (Kapp approximately 10(20)) but reversible binding of iron. These bilobate molecules have two binding sites, one per lobe, each housing one Fe3+ and the synergistic CO3(2-) ion. Crystallographic studies of human lactoferrin and rabbit serum transferrin in their iron-bound forms have characterized their binding sites and protein structure. Physical studies show that a substantial conformational change accompanies iron binding and release. We have addressed this phenomenon through crystal structure analysis of human apolactoferrin at 2.8 A resolution. In this structure the N-lobe binding cleft is wide open, following a domain rotation of 53 degrees, mediated by the pivoting of two helices and flexing of two interdomain polypeptide strands. Remarkably, the C-lobe cleft is closed, but unliganded. These observations have implications for transferrin function and for binding proteins in general.

No abstract

The three-dimensional structure of human milk lactoferrin, a member of the transferrin family, has been determined crystallographically at 3.2-resolution. The molecule has two-fold internal homology. The N-and C-terminal halves form two separate globular lobes, connected by a short a-helix, and carry one iron-binding site each. Each lobe has the same folding, based on two domains of similar supersecondary structure, with the iron site at the domain interface. Each iron atom is coordinated by four protein ligands: two tyrosines, one histidine, and one aspartate. A probable CO2-(or HCO5) ion is suggested by the electron density, bound to iron and adjacent to an arginine side chain and a helix N terminus. The protein folding and location of the binding sites show marked similarities with those of other binding proteins, notably the sulfatebinding protein from Salmonella typhimurium.Lactoferrin [also known as lactotransferrin (1)] is a member of the family of iron-binding proteins that also includes transferrin and ovotransferrin (2,3). These proteins are widely distributed in the physiological fluids of vertebrates. Although they have been the subject of intensive investigation over many years no definitive three-dimensional structural information has hitherto been available. All are monomeric glycoproteins with =700 amino acid residues and molecular weight -80,000. Each binds reversibly two iron atoms (as Fe3"), concomitantly with two C02- (or HCO ) ions. Notable features of their binding properties are (i) the synergistic relationship between cation and anion binding (4), (ii) the extremely tight binding of iron (binding constant 1020 for lactoferrin), and (iii) the fact that this tightly bound iron is nevertheless available in vivo, apparently through binding at specific receptors (5, 6).The known or proposed biological functions ofthe transferrins depend on their iron-binding properties. Thus serum transferrin, the iron transport protein in plasma, provides an iron source for hemoglobin synthesis and other metabolic requirements. Lactoferrin, widely distributed through many exocrine secretions, notably milk, and an important component of leukocytes, has strong bacteriostatic properties (7). These result from its avidity for iron, depriving bacteria of iron essential for growth. It may also protect cells from free radical damage by binding potentially catalytic free iron (8).All three proteins have bilobal structures. This is indicated by (i) fragmentation studies (ref. 9 and references therein), which demonstrate that the polypeptide chain can be cleaved into two halves, each carrying one iron site, and (ii) lowresolution x-ray studies (10). Amino acid sequence alignments (11) show that, in addition to the extensive homology between different transferring, each also shows strong twofold internal homology, indicative of gene duplication from a one-iron 40,000 molecular weight precursor molecule. For lactoferrin, there is =40% sequence identity between its Nand C-terminal halves.Here we report the resu...

a b s t r a c tO-glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine b-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC 50 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22.

Transition states can be predicted from an enzyme's affinity to related transition-state analogues. 5'-Methylthioadenosine nucleosidases (MTANs) are involved in bacterial quorum sensing pathways and thus are targets for antibacterial drug design. The transition-state characteristics of six MTANs are compared by analyzing dissociation constants (K(d)) with a small array of representative transition-state analogues. These inhibitors mimic early or late dissociative transition states with K(d) values in the picomolar range. Our results indicate that the K(d) ratio for mimics of early and late transition states are useful in distinguishing between these states. By this criterion, the transition states of Neisseria meningitides and Helicobacter pylori MTANs are early dissociative, whereas Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs have late dissociative characters. This conclusion is confirmed independently by the characteristic [1'- (3)H] and [1'- (14)C] kinetic isotope effects (KIEs) of these enzymes. Large [1'- (3)H] and unity [1'- (14)C] KIEs are observed for late dissociative transition states, whereas early dissociative states showed close-to-unity [1'- (3)H] and significant [1'- (14)C] KIEs. K d values of various MTANs for individual transition-state analogues provide tentative information about transition-state structures due to varying catalytic efficiencies of enzymes. Comparing K d ratios for mimics of early and late transition states removes limitations inherent to the enzyme and provides a better predictive tool in discriminating between possible transition-state structures.

No abstract

Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpM-TAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpM-TAN and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC90 value of < 8 ng/ml for H. pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.