Mechanism of action of nucleoside antibacterial natural product antibiotics

This article reviews the structures and biological activities of several classes of uridine-containing nucleoside antibiotics (tunicamycins, mureidomycins/pacidamycins/sansanmycins, liposidomycins/caprazamycins, muraymycins, capuramycins) that target translocase MraY on the peptidoglycan biosynthetic pathway. In particular, recent advances in structure-function studies, and recent X-ray crystal structures of translocase MraY complexed with muraymycin D2 and tunicamycin are described. The inhibition of other phospho-nucleotide transferase enzymes related to MraY by nucleoside antibiotics and analogues is also reviewed

Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide (TMAO) by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate inter-subunit electron transfer between the Rieske centre and the catalytic mononuclear iron centre. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Together, our study reveals the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assigns the role of this novel group of Rieske-type proteins in microbial carnitine metabolism

Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development

Identification of an extracellular bacterial flavoenzyme that can prevent re-polymerisation of lignin fragments

A significant problem in the oxidative breakdown of lignin is the tendency of phenolic radical fragments to re-polymerise to form higher molecular weight species. In this paper we identify an extracellular flavin-dependent dehydrolipoamide dehydrogenase from Thermobifida fusca that prevents oxidative dimerization of a dimeric lignin model compound, which could be used as an accessory enzyme for lignin depolymerisation

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage <phi>X174 lysis protein E, and cationic antibacterial peptides

This review covers recent developments in the inhibition of translocase MraY and related phospho-GlcNAc transferases WecA and TagO, and insight into the inhibition and catalytic mechanism of this class of integral membrane proteins from the structure of Aquifex aeolicus MraY. Recent studies have also identified a protein-protein interaction site in E. coli MraY, that is targeted by bacteriophage X174 lysis protein E, and also by cationic antimicrobial peptides containing Arg-Trp close to their N- or C-termini

Biocatalytic conversion of lignin to aromatic dicarboxylic acids in Rhodococcus jostii RHA1 by re-routing aromatic degradation pathways

The heteropolymer lignin represents an untapped resource for production of renewable aromatic chemicals, if efficient depolymerisation methods can be developed. In this work, the metabolic pathways in Rhodococcus jostii RHA1 for degradation of aromatic lignin breakdown products are re-routed, in order to generate an aromatic dicarboxylic acid product that could be used for bioplastic synthesis. Protocatechuic acid is normally metabolised via ortho-cleavage to the β-keto-adipate pathway. Insertion of recombinant genes for protocatechuate 4,5-dioxygenase or protocatechuate 2,3-dioxygenase into R. jostii RHA1, followed by ammonia cyclisation of the extradiol cleavage products, generates pyridine 2,4-dicarboxylic acid or pyridine 2,5-dicarboxylic acid bioproducts in yields of 80–125 mg L−1 when grown on minimal media containing 1% wheat straw lignocellulose

Structure of thermobifida fusca DyP-type peroxidase and activity towards kraft lignin and lignin model compounds

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at max 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a -aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS

Enhanced biocatalytic degradation of lignin using combinations of lignin-degrading enzymes and accessory enzymes

Methods for screening combinations of lignin-degrading enzymes and accessory enzymes for product release from polymeric lignin have been developed, using two colorimetric assays that can be applied in microtiter plate format. A set of 3 bacterial DyP-type peroxidase enzymes from Pseudomonas fluorescens, Comamonas testosteroni and Agrobacterium sp., two bacterial multi-copper oxidase enzymes CueO from Ochrobactrum sp. and CopA from Pseudomonas putida, and Sphingobacterium sp. T2 manganese superoxide dismutase have been tested in combination with one LigE β-etherase enzyme from Agrobacterium sp., two dihydrolipoamide dehydrogenase enzymes from Sphingobacterium sp. T2, Burkholderia cenocepacia peroxiredoxin, and Desulfitobacterium hafniense arylsulfotransferase. Combinations of Agrobacterium LigE with DyP-type peroxidases gave 4–10 enhancement in low molecular weight product release from technical lignins, and enhancements in product release were observed for all lignins tested, using different accessory enzymes. Analysis of products formed by reverse phase HPLC verified increases in concentrations of specific low molecular weight products

Esterase EstK from Pseudomonas putida mt-2 : an enantioselective acetylesterase with activity for deacetylation of xylan and poly(vinylacetate)

An extracellular esterase gene estK was identified in Pseudomonas putida mt-2, and was overexpressed to high levels in Escherichia coli. The recombinant EstK enzyme was purified and characterised kinetically against p-nitrophenyl ester and other aryl-alkyl ester substrates, and was found to be selective for hydrolysis of acetyl ester substrates, with high activity for p-nitrophenyl acetate (kcat 5.5 s-1, KM 285 µM). Recombinant EstK was found to catalyse deacetylation of acetylated beech xylan, indicating a possible in vivo function for this enzyme, and partial deacetylation of a synthetic polymer, poly(vinylacetate). EstK was found to catalyse enantioselective hydrolysis of racemic 1-phenylethyl acetate, generating 1R-phenylethanol with an enantiomeric excess of 80.4%

O2-independent demethylation of trimethylamine N-oxide by Tdm of Methylocella silvestris

Bacterial trimethylamine N-oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen-independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, sitedirected mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn2+ and 1 molar equivalent of non-heme Fe2+. We further investigated Zn2+ and Fe2+-binding sites through homology modelling and sitedirected mutagenesis and found that Zn2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe2+ and Zn2+ centres, either directly or indirectly via H-bonding through a neighbouring H2O molecule. H276 contributes to Fe2+ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation- interaction. Furthermore, a cross-over experiment using a substrate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn2+ and Fe2+ in the catalysis of TMAO demethylation by this unique oxygenindependent enzyme