EMA and EFSA Joint Scientific Opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the European Union, and the resulting impacts on food safety (RONAFA).

EFSA and EMA have jointly reviewed measures taken in the EU to reduce the need for and use of antimicrobials in food-producing animals, and the resultant impacts on antimicrobial resistance (AMR). Reduction strategies have been implemented successfully in some Member States. Such strategies include national reduction targets, benchmarking of antimicrobial use, controls on prescribing and restrictions on use of specific critically important antimicrobials, together with improvements to animal husbandry and disease prevention and control measures.

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes.

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics.

Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms.

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli.

Purification and characterization of actinomycin synthetase I, a 4-methyl-3-hydroxyanthranilic acid-AMP ligase from Streptomyces chrysomallus.

Actinomycin synthetase I was purified to homogeneiety from actinomycin-producing Streptomyces chrysomallus. The purified enzyme is a single polypeptide chain of M(r) 45,000. It catalyzes the formation of the adenylate of 4-methyl-3-hydroxyanthranilic acid (4-MHA) from the free acid and ATP in an equilibrium reaction.

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form.

Biosynthesis of quinoxaline antibiotics: purification and characterization of the quinoxaline-2-carboxylic acid activating enzyme from Streptomyces triostinicus.

A quinoxaline-2-carboxylic acid activating enzyme was purified to homogeneity from triostin-producing Streptomyces triostinicus. It could also be purified from quinomycin-producing Streptomyces echinatus. Triostins and quinomycins are peptide lactones that contain quinoxaline-2-carboxylic acid as chromophoric moiety.

Chromophore activating enzyme involved in the biosynthesis of the mikamycin B antibiotic etamycin from Streptomyces griseoviridus.

A 3-hydroxypicolinic acid activating enzyme from etamycin producing Streptomyces griseoviridus has been purified to apparent homogeneity. Etamycin is a member of mikamycin B antibiotics, chromopeptide lactones, which contain 3-hydroxypicolinic acid (3-HPA) as the chromophoric group. The enzyme catalyzes both the 3-HPA-dependent ATP-pyrophosphate exchange and the formation of 3-HPA adenylate from 3-HPA and ATP.

Proteinchemical and kinetic features of gramicidin S synthetase.

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10.

Formation of D-Phe-Pro-Val-cyclo-Orn by gramicidin S synthetase in the absence of L-leucine.

The preparation of both enzymes of gramicidin S synthetase was efficiently improved by introduction of the fast protein liquid chromatography technique. High-resolution anion-exchange chromatography on Pharmacia Mono Q HR 5/5 was used as the final purification step. D-Phe-Pro-Val-cyclo-Orn was obtained as a product of the multienzyme by omission of L-leucine from the complete bioassay mixture.

Reactive sulfhydryl groups involved in the aminoacyl adenylate activation reactions of the gramicidin S synthetase 2.

Six accessible sulfhydryl groups of the light component of gramicidin S synthetase (GS 1) were titrated with N-ethylmaleimide (Ma1NEt) as well as 3,3'-dithiobis(6-nitrobenzoic acid) (Nbs2). Twenty-four thiols were detected in the heavy enzyme (GS 2) using Ma1NEt as the modifier. Substrate amino acid-induced protection of GS 2 against deactivation by Ma1NEt indicates that in addition to the specific thiols at the thiotemplates of gramicidin S synthetase reactive SH groups are also involved in the primary aminoacyl adenylate activation reactions.

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone.

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents.