Resorculins: hybrid polyketide macrolides from Streptomyces sp. MST-91080.

Fourteen-membered macrolides are a class of compounds with significant clinical value as antibacterial agents. As part of our ongoing investigation into the metabolites of Streptomyces sp. MST-91080, we report the discovery of resorculins A and B, unprecedented 3,5-dihydroxybenzoic acid (α-resorcylic acid)-containing 14-membered macrolides. We sequenced the genome of MST-91080 and identified the putative resorculin biosynthetic gene cluster (rsn BGC). The rsn BGC is hybrid of type I and type III polyketide synthases. Bioinformatic analysis revealed that the resorculins are relatives of known hybrid polyketides: kendomycin and venemycin. Resorculin A exhibited antibacterial activity against Bacillus subtilis (MIC 19.8 µg mL-1), while resorculin B showed cytotoxic activity against the NS-1 mouse myeloma cell line (IC50 3.6 µg mL-1).

Recently Discovered Secondary Metabolites from Streptomyces Species.

The Streptomyces genus has been a rich source of bioactive natural products, medicinal chemicals, and novel drug leads for three-quarters of a century. Yet studies suggest that the genus is capable of making some 150,000 more bioactive compounds than all Streptomyces secondary metabolites reported to date. Researchers around the world continue to explore this enormous potential using a range of strategies including modification of culture conditions, bioinformatics and genome mining, heterologous expression, and other approaches to cryptic biosynthetic gene cluster activation. Our survey of the recent literature, with a particular focus on the year 2020, brings together more than 70 novel secondary metabolites from Streptomyces species, which are discussed in this review. This diverse array includes cyclic and linear peptides, peptide derivatives, polyketides, terpenoids, polyaromatics, macrocycles, and furans, the isolation, chemical structures, and bioactivity of which are appraised. The discovery of these many different compounds demonstrates the continued potential of Streptomyces as a source of new and interesting natural products and contributes further important pieces to the mostly unfinished puzzle of Earth's myriad microbes and their multifaceted chemical output.

Yeppoonic acids A - D: 1,2,4-trisubstituted arene carboxylic acid co-metabolites of conglobatin from an Australian Streptomyces sp.

Streptomyces sp. MST-91080 was isolated from a soil sample collected in Queensland, Australia. From this strain, yeppoonic acids A - D were purified and spectroscopically characterised. The yeppoonic acids are a family of diene enecarboxylic acids on a 1,2,4-trisubstituted benzene scaffold, structurally related to other Streptomyces secondary metabolites MF-EA-705α/ß, NFAT-133 and the lorneic acids. Yeppoonic acids B and C show strong cytotoxicity against the NS-1 mouse myeloma cell line (IC50 2.3 µg ml-1 and 3.8 µg ml-1, respectively) and moderate activity against the DU 145 human prostate cancer cell line (IC50 32.8 µg ml-1 and 49.6 µg ml-1, respectively).

Isopenicillin†N Synthase: Crystallographic Studies.

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase (NHIO) that catalyses the cyclisation of tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN). Over the last 25 years, crystallography has shed considerable light on the mechanism of IPNS catalysis. The first crystal structure, for apo-IPNS with Mn bound in place of Fe at the active site, reported in 1995, was also the first structure for a member of the wider NHIO family. This was followed by the anaerobic enzyme-substrate complex IPNS-Fe-ACV (1997), this complex plus nitric oxide as a surrogate for co-substrate dioxygen (1997), and an enzyme product complex (1999). Since then, crystallography has been used to probe many aspects of the IPNS reaction mechanism, by crystallising the protein with a diversity of substrate analogues and triggering the oxidative reaction by using elevated oxygen pressures to force the gaseous co-substrate throughout protein crystals and maximise synchronicity of turnover in crystallo. In this way, X-ray structures have been elucidated for a range of complexes closely related to and/or directly derived from key intermediates in the catalytic cycle, thereby answering numerous mechanistic questions that had arisen from solution-phase experiments, and posing many new ones. The results of these crystallographic studies have, in turn, informed computational experiments that have brought further insight. These combined crystallographic and computational investigations augment and extend the results of earlier spectroscopic analyses and solution phase studies of IPNS turnover, to enrich our understanding of this important protein and the wider NHIO enzyme family.

Conglobatins B-E: cytotoxic analogues of the C2-symmetric macrodiolide conglobatin.

Chemical investigation of a previously unreported indigenous Australian Streptomyces strain MST-91080 has identified six novel analogues related to the oxazole-pendanted macrodiolide, conglobatin. Phylogenetic analysis of the 16S rRNA gene sequence identified MST-91080 as a species of Streptomyces, distinct from reported conglobatin producer, Streptomyces conglobatus ATCC 31005. Conglobatins B-E diverge from conglobatin through differing patterns of methylation on the macrodiolide skeleton. The altered methyl positions suggest a deviation from the published biosynthetic pathway, which proposed three successive methylmalonyl-CoA extender unit additions to the conglobatin monomer. Conglobatins B1, C1 and C2 exhibited more potent cytotoxic activity selectively against the NS-1 myeloma cell line (IC50 0.084, 1.05 and 0.45 µg ml-1, respectively) compared with conglobatin (IC50 1.39 µg ml-1).

Metal complexes as a promising source for new antibiotics.

There is a dire need for new antimicrobial compounds to combat the growing threat of widespread antibiotic resistance. With a currently very scarce drug pipeline, consisting mostly of derivatives of known antibiotics, new classes of antibiotics are urgently required. Metal complexes are currently in clinical development for the treatment of cancer, malaria and neurodegenerative diseases. However, only little attention has been paid to their application as potential antimicrobial compounds. We report the evaluation of 906 metal-containing compounds that have been screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD) for antimicrobial activity. Metal-bearing compounds display a significantly higher hit-rate (9.9%) when compared to the purely organic molecules (0.87%) in the CO-ADD database. Out of 906 compounds, 88 show activity against at least one of the tested strains, including fungi, while not displaying any cytotoxicity against mammalian cell lines or haemolytic properties. Herein, we highlight the structures of the 30 compounds with activity against Gram-positive and/or Gram-negative bacteria containing Mn, Co, Zn, Ru, Ag, Eu, Ir and Pt, with activities down to the nanomolar range against methicillin resistant S. aureus (MRSA). 23 of these complexes have not been reported for their antimicrobial properties before. This work reveals the vast diversity that metal-containing compounds can bring to antimicrobial research. It is important to raise awareness of these types of compounds for the design of truly novel antibiotics with potential for combatting antimicrobial resistance.

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed ß-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl ß-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl ß-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general.

Discovery of microbial natural products by activation of silent biosynthetic gene clusters.

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities and a wide variety of applications in medicine and agriculture, such as the treatment of infectious diseases and cancer, and the prevention of crop damage. Genomics has revealed that many microorganisms have far greater potential to produce specialized metabolites than was thought from classic bioactivity screens; however, realizing this potential has been hampered by the fact that many specialized metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures. In this Review, we discuss the strategies that have been developed in bacteria and fungi to identify and induce the expression of such silent BGCs, and we briefly summarize methods for the isolation and structural characterization of their metabolic products.

Iron complexes of tetramine ligands catalyse allylic hydroxyamination via a nitroso-ene mechanism.

Iron(II) complexes of the tetradentate amines tris(2-pyridylmethyl)amine (TPA) and N,N'-bis(2-pyridylmethyl)-N,N'-dimethylethane-1,2-diamine (BPMEN) are established catalysts of C-O bond formation, oxidising hydrocarbon substrates via hydroxylation, epoxidation and dihydroxylation pathways. Herein we report the capacity of these catalysts to promote C-N bond formation, via allylic amination of alkenes. The combination of N-Boc-hydroxylamine with either FeTPA (1 mol %) or FeBPMEN (10 mol %) converts cyclohexene to the allylic hydroxylamine (tert-butyl cyclohex-2-en-1-yl(hydroxy)carbamate) in moderate yields. Spectroscopic studies and trapping experiments suggest the reaction proceeds via a nitroso-ene mechanism, with involvement of a free N-Boc-nitroso intermediate. Asymmetric induction is not observed using the chiral tetramine ligand (+)-(2R,2'R)-1,1'-bis(2-pyridylmethyl)-2,2'-bipyrrolidine ((R,R')-PDP).

Bio-inspired nitrile hydration by peptidic ligands based on L-cysteine, L-methionine or L-penicillamine and pyridine-2,6-dicarboxylic acid.

Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme which catalyses the conversion of nitriles to amides. The high efficiency and broad substrate range of NHase have led to the successful application of this enzyme as a biocatalyst in the industrial syntheses of acrylamide and nicotinamide and in the bioremediation of nitrile waste. Crystal structures of both cobalt(III)- and iron(III)-dependent NHases reveal an unusual metal binding motif made up from six sequential amino acids and comprising two amide nitrogens from the peptide backbone and three cysteine-derived sulfur ligands, each at a different oxidation state (thiolate, sulfenate and sulfinate). Based on the active site geometry revealed by these crystal structures, we have designed a series of small-molecule ligands which integrate essential features of the NHase metal binding motif into a readily accessible peptide environment. We report the synthesis of ligands based on a pyridine-2,6-dicarboxylic acid scaffold and L-cysteine, L-S-methylcysteine, L-methionine or L-penicillamine. These ligands have been combined with cobalt(III) and iron(III) and tested as catalysts for biomimetic nitrile hydration. The highest levels of activity are observed with the L-penicillamine ligand which, in combination with cobalt(III), converts acetonitrile to acetamide at 1.25 turnovers and benzonitrile to benzamide at 1.20 turnovers.

Incorporating a piperidinyl group in the fluorophore extends the fluorescence lifetime of click-derived cyclam-naphthalimide conjugates.

Ligands incorporating a tetraazamacrocycle receptor, a 'click'-derived triazole and a 1,8-naphthalimide fluorophore have proven utility as probes for metal ions. Three new cyclam-based molecular probes are reported, in which a piperidinyl group has been introduced at the 4-position of the naphthalimide fluorophore. These compounds have been synthesized using the copper(I)-catalyzed azide-alkyne Huisgen cycloaddition and their photophysical properties studied in detail. The alkylamino group induces the expected red-shift in absorption and emission spectra relative to the simple naphthalimide derivatives and gives rise to extended fluorescence lifetimes in aqueous buffer. The photophysical properties of these systems are shown to be highly solvent-dependent. Screening the fluorescence responses of the new conjugates to a wide variety of metal ions reveals significant and selective fluorescence quenching in the presence of copper(II), yet no fluorescence enhancement with zinc(II) as observed previously for the simple naphthalimide derivatives. Reasons for this different behaviour are proposed. Cytotoxicity testing shows that these new cyclam-triazole-dye conjugates display little or no toxicity against either DLD-1 colon carcinoma cells or MDA-MB-231 breast carcinoma cells, suggesting a potential role for these and related systems in biological sensing applications.

The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site.

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of ß-lactam antibiotics. IPNS converts the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(L-α-aminoadipoyl)-L-cysteinyl-O-methyl-D-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(L-α-aminoadipoyl)-L-cysteinyl-O-methyl-D-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the ß-carbon atom of their third residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue.

The interaction of isopenicillin N synthase with homologated substrate analogues δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-Xaa characterised by protein crystallography.

Isopenicillin N synthase (IPNS) converts the linear tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution-phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-valine (AhCV) and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-allylglycine (AhCaG) into monocyclic hydroxy-lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution-phase results with other tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-Xaa, we have crystallised AhCV and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-S-methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:Fe(II):AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:Fe(II):AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures.

The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue.

Isopenicillin N synthase (IPNS) converts its linear tripeptide substrate δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to bicyclic isopenicillin N (IPN), the key step in penicillin biosynthesis. Solution-phase incubation experiments have shown that IPNS will accept and oxidise a diverse array of substrate analogues, including tripeptides that incorporate L-homocysteine as their second residue, and tripeptides with truncated side-chains at the third amino acid such as δ-L-α-aminoadipoyl-L-cysteinyl-D-α-aminobutyrate (ACAb), δ-L-α-aminoadipoyl-L-cysteinyl-D-alanine (ACA) and δ-L-α-aminoadipoyl-L-cysteinyl-glycine (ACG). However IPNS does not react with dipeptide substrates. To probe this selectivity we have crystallised the enzyme with the dipeptide δ-L-α-aminoadipoyl-L-homocysteine (AhC) and solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 Å resolution. This structure reveals an unexpected mode of peptide binding at the IPNS active site, in which the homocysteinyl thiolate does not bind to iron. Instead the primary mode of binding sees the homocysteinyl carboxylate coordinated to the metal, while its side-chain is oriented into the region of the active site normally occupied by the benzyl group of protein residue Phe211.

Investigating the oxidation of alkenes by non-heme iron enzyme mimics.

Iron is emerging as a key player in the search for efficient and environmentally benign methods for the functionalisation of C-H bonds. Non-heme iron enzymes catalyse a diverse array of oxidative chemistry in nature, and small-molecule complexes designed to mimic the non-heme iron active site have great potential as C-H activation catalysts. Herein we report the synthesis of a series of organic ligands that incorporate key features of the non-heme iron active site. Iron(II) complexes of these ligands have been generated in situ and their ability to promote hydrocarbon oxidation has been investigated. Several of these systems promote the biomimetic dihydroxylation of cyclohexene at low levels, when hydrogen peroxide is used as the oxidant; allylic oxidation products are also observed. An investigation of ligand stability reveals formation of several breakdown products under the conditions of the oxidative turnover reactions. These products arise via oxidative decarboxylation, dehydration and deamination reactions. Taken together these results indicate that competing mechanisms are at play with these systems: biomimetic hydroxylation involving high-valent iron species, and allylic oxidation via Fenton chemistry and Haber-Weiss radical pathways.

Copper, nickel, and zinc cyclam-amino acid and cyclam-peptide complexes may be synthesized with "click" chemistry and are noncytotoxic.

We describe the synthesis of cyclam metal complexes derivatized with amino acids or a tripeptide using a copper(I)-catalyzed Huisgen "click" reaction. The linker triazole formed during the synthesis plays an active coordinating role in the complexes. The reaction conditions do not racemize the amino acid stereocenters. However, a methylene group adjacent to the triazole is susceptible to H/D exchange under ambient conditions, an observation which has potentially important implications for structures involving stereocenters adjacent to triazoles in click-derived structures. The successful incorporation of several amino acids is described, including reactive tryptophan and cysteine side chains. All complexes are formed rapidly upon introduction of the relevant metal salt, including synthetically convenient cases where trifluoroacetate salts of cyclam derivatives are used directly in the metalation. None of the metal complexes displayed any cytotoxicity to mammalian cells, suggesting that the attachment of such complexes to amino acids and peptides does not induce toxicity, further supporting their potential suitability for labeling/imaging studies. One Cu(II)-cyclam-triazole-cysteine disulfide complex displayed moderate activity against MCF-10A breast nontumorigenic epithelial cells.

The crystal structure of isopenicillin N synthase with δ-((L)-α-aminoadipoyl)-(L)-cysteinyl-(D)-methionine reveals thioether coordination to iron.

Isopenicillin N synthase (IPNS) catalyses cyclization of δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS turns over a wide range of substrate analogues in which the valine residue of its natural substrate is replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon sidechains in this position, however the enzyme is less tolerant of analogues presenting polar functionality in place of the valinyl isopropyl group. We report a new ACV analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been elucidated for the IPNS:Fe(II):ACM complex at 1.40Å resolution. This structure reveals that ACM binds in the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57Å. The sulfur of the cysteinyl thiolate sits 2.36Å from the metal.

Isopenicillin N synthase binds δ-(L-α-aminoadipoyl)-L-cysteinyl-D-thia-allo-isoleucine through both sulfur atoms.

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution-phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ-L-α-aminoadipoyl-L-cysteinyl-D-isoleucine (ACI) and AC-D-allo-isoleucine (ACaI) to penam products, the isosteric sulfur-containing peptides AC-D-thiaisoleucine (ACtI) and AC-D-thia-allo-isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe(II) :ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.

Boronated phosphonium salts containing arylboronic acid, closo-carborane, or nido-carborane: synthesis, X-ray diffraction, in vitro cytotoxicity, and cellular uptake.

The preparation of boronated triaryl and tetraaryl phosphonium salts of the type [PPh(3)CH(2)R]Br [R is 4-boronophenyl (1), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (2), 3-boronophenyl (3), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (4), 2-boronophenyl (5), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-yl)phenyl (6), and closo-1,2-carboran-1-yl (7)] is described. These compounds were prepared by the reaction of triphenylphosphine with benzylic bromides or 1-bromomethyl-closo-1,2-carborane in acetonitrile solution at 85 °C. The zwitterionic nido-7,8-carborane derivative PPh(3)CH(2)C(2)B(9)H(11) (8) was prepared by treatment of 7 with cesium fluoride in refluxing ethanol. All compounds were fully characterized by multinuclear ((1)H, (11)B, (13)C, and (31)P) 1D- and 2D-NMR spectroscopy, electrospray ionization mass spectrometry, and elemental analysis, and single-crystal X-ray structures were determined for compounds 1, 3, 7, and 8. The cytotoxicities and boron uptake of selected derivatives were investigated in vitro using human glioblastoma (T98G) and canine kidney tubule (MDCK II) cells. The zwitterionic species 8 was found to be the least cytotoxic agent while also delivering the greatest amount of boron to the T98G cells, peaking at 9.15 ± 2.65 µg B/mg protein.

Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-valine (lld-ACV). Herein we report crystallographic studies to investigate the binding of a truncated lll-substrate in the active site of IPNS. Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with the enzyme. delta-l-alpha-Aminoadipoyl-l-cysteinyl-d-2-amino-3,3-dideuteriobutyrate (lld-ACd(2)Ab) has the same configuration as the natural substrate lld-ACV at each of its three stereocentres; its epimer delta-l-alpha-aminoadipoyl-l-cysteinyl-l-2-amino-3,3-dideuteriobutyrate (lll-ACd(2)Ab) has the opposite configuration at its third amino acid. lll-ACV has previously been shown to inhibit IPNS turnover of its substrate lld-ACV; the all-protiated tripeptide delta-l-alpha-aminoadipoyl-l-cysteinyl-d-2-aminobutyrate (lld-ACAb) is a substrate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the crystal structures of the IPNS:Fe(II):lld-ACd(2)Ab and IPNS:Fe(II):lll-ACd(2)Ab complexes offer a possible rationale for the previously observed inhibitory effects of lll-ACV on IPNS activity.