Hybrid nonribosomal peptide-polyketide interfaces in epothilone biosynthesis: minimal requirements at N and C termini of EpoB for elongation.

Epothilone (Epo) D, an antitumor agent currently in clinical trials, is a hybrid natural product produced by the combined action of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). In the epothilone biosynthetic pathway, EpoB, a 165 kDa NRPS is inserted into an otherwise entirely PKS assembly line, forming two hybrid NRPS-PKS interfaces. In light of the terminal linker effect previously identified in PKS, the N- and C-terminal sequences of EpoB were examined for their roles in propagating the incipient natural product. Eight amino acid residues at EpoB C terminus, in which six are positively charged, were found to be a key component of the C-terminal linker effect. A minimal sequence of 56 residues at EpoB N terminus was required for elongating the acetyl group from the acyl carrier protein (ACP) of EpoA to form methylthiazolyl-S-EpoB.

Two-peptide bacteriocins produced by lactic acid bacteria.

Bacteriocins from lactic acid bacteria are ribosomally produced peptides (usually 30-60 amino acids) that display potent antimicrobial activity against certain other Gram-positive organisms. They function by disruption of the membrane of their targets, mediated in at least some cases by interaction of the peptide with a chiral receptor molecule (e.g., lipid II or sugar PTS proteins). Some bacteriocins are unmodified (except for disulfide bridges), whereas others (i.e. lantibiotics) possess extensive post-translational modifications which include multiple monosulfide (lanthionine) bridges and dehydro amino acids as well as possible keto amide residues at the N-terminus. Most known bacteriocins are biologically active as single peptides. However, there is a growing class of two peptide systems, both unmodified and lantibiotic, which are fully active only when both partners are present (usually 1:1). In some cases, neither peptide has activity by itself, whereas in others, the activity of one is enhanced by the other. This review discusses the classification, structure, production, regulation, biological activity, and potential applications of such two-peptide bacteriocins.

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis.

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k(cat) of 1.4 min(-1) was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.

Characterization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis.

The aminocoumarin antibiotics clorobiocin and coumermycin A(1) target the B subunit of DNA gyrase by presentation of the 5-methyl-pyrrolyl-2-carboxy ester moiety in the ATP-binding site of the enzyme. The pyrrolyl pharmacophore is derived by a four electron oxidation of a prolyl unit while tethered in phosphopantetheinyl thioester linkage to a peptidyl carrier protein (PCP) subunit. l-Proline is selected and activated as l-prolyl-AMP by adenylation domain enzymes (CloN4 and CouN4) and then installed as the thioester on the holo form of the PCP proteins CloN5 and CouN5. Enzymatic oxidation of the prolyl-S-PCP by the flavoprotein dehydrogenase CloN3 can be followed by rapid quench and subsequent electrospray ionization-Fourier transform mass spectrometry analysis of the acyl-S-protein substrate/product mixture to establish that a two-electron oxidized pyrrolinyl-S-enzyme transiently accumulates on the way to the four-electron oxidized, heteroaromatic pyrrolyl-2-carboxy-S-PCP acyl enzyme product.

Synthesis of mono- and disaccharide analogs of moenomycin and lipid II for inhibition of transglycosylase activity of penicillin-binding protein 1b.

Three types of mono- and disaccharides 3a,b, 4a-c, 5, and some chaetomellic acid A analogs 6 and 42-44 were synthesized as potential inhibitors of the transglycosylase activity of penicillin-binding protein 1b (PBP1b), a key bacterial enzyme responsible for the formation of the polysaccharide backbone of peptidoglycan as well as for cross-linking of its peptide portions. The target compounds combine structural features of both the active portion of moenomycin and the natural PBP1b substrate, lipid II. The desired skeletons were obtained in a convergent fashion involving attachment of the lipid-alkylated glyceric acid moieties 11a,b to the corresponding carbohydrate-containing phosphonic acids 23, 24a, and 24b. Compounds 3a,b were prepared to verify the distance requirements between the sugar and the noncleavable C-phosphonate moieties. Compounds 4a-c were synthesized to examine the importance of the first sugar unit of moenomycin, a known inhibitor of transglycosylase catalysis by PBP1b, with respect to antibiotic activity. These were prepared by condensation of 11a,b with 28a and 28c, which were made by glycosylation of 3-bromopropanol with oxazolines 25a,b, and Arbuzov reaction with triethyl or trimethyl phosphite, followed by dealkylation with bromotrimethylsilane. Compound 5 was generated to verify the possibility of using a dicarboxylate group to mimic the diphosphate of lipid II. It was synthesized by coupling of alcohol 31 with alpha-trichloroacetimidate 34. Chaetomellic acid A analogs were prepared by a Michael addition to dimethyl acetylenedicarboxylate. With the exception of 3b, all of the target compounds were found to inhibit PBP1b, albeit with modest potency.

Purification and characterization of brochocin A and brochocin B(10-43), a functional fragment generated by heterologous expression in Carnobacterium piscicola.

Brochothrix campestris ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of Clostridium botulinum. An improved purification method was developed for BrcC, in which n-butanol and chloroform extraction are used. Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by B. campestris as mature peptides consisting of 59 and 43 amino acids, respectively. Separate expression clones of BrcA and BrcB were constructed previously in Carnobacterium piscicola LV17C, but the products were not chemically characterized. Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by C. piscicola as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site. This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex. Circular dichroism measurements revealed a high beta-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture. Separate expression clones of brcA and brcB were also constructed in Escherichia coli, but these clones only produced multiple fragments of the desired peptides with little or no activity.

Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase.

Rubber transferase, a cis-prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4-7) and alpha-hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of compounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species-specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes.