Targeted modifications of neomycin and paromomycin: Towards resistance-free antibiotics?

Despite their clinical importance, saving numerous human lifes, over- and mis-uses of antibiotics have created a strong selective pressure on bacteria, which induces the emergence of (multi)resistant strains. Antibioresistance is becoming so pregnant that since 2017, WHO lists bacteria threatening most human health (AWaRe, ESKAPE lists), and those for which new antibiotics are urgently needed. Since the century turn, this context is leading to a burst in the chemical synthesis of new antibiotics, mostly derived from natural antibiotics. Among them, aminoglycosides, and especially the neomycin family, exhibit broad spectrum of activity and remain clinically useful drugs. Therefore, numerous endeavours have been undertaken to modify aminoglycosides with the aim of overcoming bacterial resistances. After having replaced antibiotic discovery into an historical perspective, briefly surveyed the aminoglycoside mode of action and the associated resistance mechanisms, this review emphasized the chemical syntheses performed on the neomycin family and the corresponding structure activity relationships in order to reveal the really efficient modifications able to convert neomycin and its analogues into future drugs. This review would help researchers to strategically design novel aminoglycoside derivatives for the development of clinically viable drug candidates.

Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

Chitosan Contribution to Therapeutic and Vaccinal Approaches for the Control of Leishmaniasis.

The control of leishmaniases, a complex parasitic disease caused by the protozoan parasite Leishmania, requires continuous innovation at the therapeutic and vaccination levels. Chitosan is a biocompatible polymer administrable via different routes and possessing numerous qualities to be used in the antileishmanial strategies. This review presents recent progress in chitosan research for antileishmanial applications. First data on the mechanism of action of chitosan revealed an optimal in vitro intrinsic activity at acidic pH, high-molecular-weight chitosan being the most efficient form, with an uptake by pinocytosis and an accumulation in the parasitophorous vacuole of Leishmania-infected macrophages. In addition, the immunomodulatory effect of chitosan is an added value both for the treatment of leishmaniasis and the development of innovative vaccines. The advances in chitosan chemistry allows pharmacomodulation on amine groups opening various opportunities for new polymers of different size, and physico-chemical properties adapted to the chosen routes of administration. Different formulations have been studied in experimental leishmaniasis models to cure visceral and cutaneous leishmaniasis, and chitosan can act as a booster through drug combinations with classical drugs, such as amphotericin B. The various architectural possibilities given by chitosan chemistry and pharmaceutical technology pave the way for promising further developments.

A structural basis for the antibiotic resistance conferred by an N1-methylation of A1408 in 16S rRNA.

The aminoglycoside resistance conferred by an N1-methylation of A1408 in 16S rRNA by a novel plasmid-mediated methyltransferase NpmA can be a future health threat. In the present study, we have determined crystal structures of the bacterial ribosomal decoding A site with an A1408m1A antibiotic-resistance mutation both in the presence and absence of aminoglycosides. G418 and paromomycin both possessing a 6'-OH group specifically bind to the mutant A site and disturb its function as a molecular switch in the decoding process. On the other hand, binding of gentamicin with a 6'-NH3+ group to the mutant A site could not be observed in the present crystal structure. These observations agree with the minimum inhibitory concentration of aminoglycosides against Escherichia coli. In addition, one of our crystal structures suggests a possible conformational change of A1408 during the N1-methylation reaction by NpmA. The structural information obtained explains how bacteria acquire resistance against aminoglycosides along with a minimum of fitness cost by the N1-methylation of A1408 and provides novel information for designing the next-generation aminoglycoside.

Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.

Liposomal formulations in the pharmacological treatment of leishmaniasis: a review.

Conventional chemotherapy for leishmaniasis includes considerably toxic drugs and reports of drug-resistance are not uncommon. Liposomal encapsulated drugs appear as an option for the treatment of leishmaniasis, providing greater efficacy for the active and reducing its side effects by promoting superior tissue absorption, favouring drug penetration into the macrophages, and retarding its clearance from the site of action. In this paper, a review on the advances achieved with liposome-based anti-leishmaniasis drug delivery systems is presented. Formulations prepared with either conventional or modified (sugar-coated, cationic, niosomes, peptides- and antibodies-bounded) liposomes for the delivery of pentavalent antimonials, amphotericin B, pentamidine, paromomycyn, and miltefosine were covered. This literature review depicts a scenario of no effective therapeutic agents for the treatment of this neglected disease, where liposomal formulations appear to improve the effectiveness of the available antileishmania agents.

Abundance and distribution of antibiotic resistance genes in a full-scale anaerobic-aerobic system alternately treating ribostamycin, spiramycin and paromomycin production wastewater.

The occurrence of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs) has been intensively investigated for wastewater treatment systems treating single class of antibiotic in recent years. However, the impacts of alternately occurring antibiotics in antibiotic production wastewater on the behavior of ARGs in biological treatment systems were not well understood yet. Herein, techniques including high-capacity quantitative PCR and quantitative PCR (qPCR) were used to investigate the behavior of ARGs in an anaerobic-aerobic full-scale system. The system alternately treated three kinds of antibiotic production wastewater including ribostamycin, spiramycin and paromomycin, which referred to stages 1, 2 and 3. The aminoglycoside ARGs (52.1-79.3%) determined using high-capacity quantitative PCR were the most abundant species in all sludge samples of the three stages. The total relative abundances of macrolide-lincosamide-streptogramin (MLS) resistance genes and aminoglycoside resistance genes measured using qPCR were significantly higher (P < 0.05) in aerobic sludge than in sewage sludge. However, the comparison of ARGs acquired from three alternate stages revealed that MLS genes and the aminoglycoside ARGs did not vary significantly (P > 0.05) in both aerobic and anaerobic sludge samples. In aerobic sludge, one acetyltransferase gene (aacA4) and the other three nucleotidyltransferase genes (aadB, aadA and aadE) exhibited positive correlations with intI1 (r 2 = 0.83-0.94; P < 0.05), implying the significance of horizontal transfer in their proliferation. These results and facts will be helpful to understand the abundance and distribution of ARGs from antibiotic production wastewater treatment systems.

Substrate-dependent switching of the allosteric binding mechanism of a dimeric enzyme.

Enzyme activity is commonly controlled by allostery, where ligand binding at one site alters the activities of distant sites. Classical explanations for multisubunit proteins involve conformational transitions that are fundamentally deterministic. For example, in the Monod-Wyman-Changeaux (MWC) paradigm, conformational transitions occur simultaneously in all subunits. In the Koshland-Nemethy-Filmer (KNF) paradigm, conformational transitions only occur in ligand-bound subunits. In contrast, recent models predict conformational changes that are governed by probabilities rather than absolute rules. To better understand allostery at the molecular level, we applied a recently developed spectroscopic and calorimetric method to the interactions of a dimeric enzyme with two different ligands. We found that conformational transitions appear MWC-like for a ligand that binds at the dimer interface and KNF-like for a distal ligand. These results provide strong experimental support for probabilistic allosteric theory predictions that an enzyme can exhibit a mixture of MWC and KNF character, with the balance partly governed by subunit interface energies.

Glucose-Nucleobase Pseudo Base Pairs: Biomolecular Interactions within DNA.

Noncovalent forces rule the interactions between biomolecules. Inspired by a biomolecular interaction found in aminoglycoside-RNA recognition, glucose-nucleobase pairs have been examined. Deoxyoligonucleotides with a 6-deoxyglucose insertion are able to hybridize with their complementary strand, thus exhibiting a preference for purine nucleobases. Although the resulting double helices are less stable than natural ones, they present only minor local distortions. 6-Deoxyglucose stays fully integrated in the double helix and its OH groups form two hydrogen bonds with the opposing guanine. This 6-deoxyglucose-guanine pair closely resembles a purine-pyrimidine geometry. Quantum chemical calculations indicate that glucose-purine pairs are as stable as a natural T-A pair.

Targeted delivery of paromomycin in murine infectious diseases through association to nano lipid systems.

Treatment of intracellular infections such as those caused by Mycobacterium spp. and Leishmania spp. is often hampered by limited access of drugs to infected cells. This is the case of paromomycin (PRM), an antibiotic with broad spectrum in vitro activity against protozoa and mycobacteria. Association of chemotherapeutics to liposomes is a worthy strategy to circumvent poor drug accessibility. Six different PRM liposomal formulations were produced, physicochemically characterized and biologically evaluated in a macrophagic cell line confirming their adequacy for in vivo studies. Biodistribution profiles of PRM liposomes revealed preferential targeting of the antibiotic to the liver, spleen and lungs, relative to free PRM, which translated into an enhanced therapeutic effect in murine models infected with Mycobacterium avium and Leishmania infantum and an absence of toxic effects. Our findings demonstrate the advantages of associating PRM to liposomes indicating their potential as an alternative therapeutic strategy for mycobacterial and parasite infections. FROM THE CLINICAL EDITOR: Infections caused by intracellular organisms such as Mycobacterium and Leishmania remain a significant problem worldwide. Although effective drugs are available, their actions are limited by access into the intracellular compartment. In this article, the authors developed different liposomal formulations as drug carriers of paromomycin and investigated their efficacy in a mouse model. The positive should provide another treatment option for these organisms in the near future.

Protein dynamics are influenced by the order of ligand binding to an antibiotic resistance enzyme.

The aminoglycoside N3 acetyltransferase-IIIb (AAC) is responsible for conferring bacterial resistance to a variety of aminoglycoside antibiotics. Nuclear magnetic resonance spectroscopy and dynamic light scattering analyses revealed a surprising result; the dynamics of the ternary complex between AAC and its two ligands, an antibiotic and coenzyme A, are dependent upon the order in which the ligands are bound. Additionally, two structurally similar aminoglycosides, neomycin and paromomycin, induce strikingly different dynamic properties when they are in their ternary complexes. To the best of our knowledge, this is the first example of a system in which two identically productive pathways of forming a simple ternary complex yield significant differences in dynamic properties. These observations emphasize the importance of the sequence of events in achieving optimal protein-ligand interactions and demonstrate that even a minor difference in molecular structure can have a profound effect on biochemical processes.

Ubiquitination-mediated interaction among domains is responsible for inhibition of RNA endonuclease activity of mRNA cycling sequence binding protein from L. donovani (LdCSBP).

In nearly complete absence of transcriptional regulation, messenger RNA (mRNA) turnover mediated through specific cis-elements plays a predominant role in the control of differential gene expression for the disease causing trypanosomatid parasites. In these organisms, the periodic accumulation of S-phase messages during cell cycle is determined by the presence of one or more copies of a conserved CAUAGAAG octanucleotide motif in the untranslated regions of mRNAs. In our previous studies, a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani was characterized, which binds specifically to the octamer-containing RNAs via its uniquely arranged CCCH-type Zn fingers and degrades them through its small MutS-related (Smr) endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Interestingly, the protein is modified by the incorporation of a monoubiquitin residue, and the posttranslational modification inhibits its riboendonuclease activity. However, the mechanism of such inhibition was previously unknown. Here, we establish that the CCCH-type Zn finger domain is the site of ubiquitination in LdCSBP and the interaction of CUE domain of the protein with the ubiquitinated Zn finger domain is responsible for inhibition of its riboendonuclease activity. The findings elucidate an inhibitory mechanism of RNA cleavage through ubiquitination-mediated intramolecular interaction among domains of the enzyme. Furthermore, the riboendonuclease activity is inhibited by anti-leishmanial drug paromomycin suggesting that the regulation of RNA metabolism could be a target of the drug.

Redesign of substrate specificity and identification of the aminoglycoside binding residues of Eis from Mycobacterium tuberculosis.

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis . Eis from M. tuberculosis (Eis_Mtb) and M. smegmatis (Eis_Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_Mtb is divided into two narrow channels, whereas Eis_Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_Mtb and for the functional differences from Eis_Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_Mtb by mutating them into their Eis_Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis_Mtb and Eis_Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_Mtb into Ala plays a role in APR acetylation.

Di-alkylated paromomycin derivatives: targeting the membranes of gram positive pathogens that cause skin infections.

A collection of paromomycin-based di-alkylated cationic amphiphiles differing in the lengths of their aliphatic chain residues were designed, synthesized, and evaluated against 14 Gram positive pathogens that are known to cause skin infections. Paromomycin derivatives that were di-alkylated with C7 and C8 linear aliphatic chains had improved antimicrobial activities relative to the parent aminoglycoside as well as to the clinically used membrane-targeting antibiotic gramicidin D; several novel derivatives were at least 16-fold more potent than the parent aminoglycoside paromomycin. Comparison between a di-alkylated and a mono-alkylated paromomycin indicated that the di-alkylation strategy leads to both an improvement in antimicrobial activity and to a dramatic reduction in undesired red blood cell hemolysis caused by many aminoglycoside-based cationic amphiphiles. Scanning electron microscopy provided evidence for cell surface damage by the reported di-alkylated paromomycins.

Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale.

Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.

Structural basis for aminoglycoside inhibition of bacterial ribosome recycling.

Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.

RNA Chemical Labeling with Site-Specific, Relative Quantification by Mass Spectrometry for the Structural Study of a Neomycin-Sensing Riboswitch Aptamer Domain.

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.

Thermodynamics of nucleic acid "shape readout" by an aminosugar.

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator displacement (FID), computational docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, the results suggest the following. (1) Neomycin binds three RNA structures [16S A site rRNA, poly(rA)·poly(rA), and poly(rA)·poly(rU)] with high affinities (K(a) ~ 10(7) M(-1)). (2) The binding of neomycin to A-form GC-rich oligomer d(A(2)G(15)C(15)T(2))(2) has an affinity comparable to those of RNA structures. (3) The binding of neomycin to DNA·RNA hybrids shows a 3-fold variance that can be attributed to their structural differences [for poly(dA)·poly(rU), K(a) = 9.4 × 10(6) M(-1), and for poly(rA)·poly(dT), K(a) = 3.1 × 10(6) M(-1)]. (4) The interaction of neomycin with DNA triplex poly(dA)·2poly(dT) yields a binding affinity (K(a)) of 2.4 × 10(5) M(-1). (5) Poly(dA-dT)(2) shows the lowest association constant for all nucleic acids studied (K(a) < 10(5)). (6) Neomycin binds to G-quadruplexes with K(a) values of ~10(4)-10(5) M(-1). (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin's affinity for various nucleic acid structures can be ranked as follows: RNAs and GC-rich d(A(2)G(15)C(15)T(2))(2) structures > poly(dA)·poly(rU) > poly(rA)·poly(dT) > T·A-T triplex, G-quadruplex, B-form AT-rich, or GC-rich DNA sequences. The results illustrate the first example of a small molecule-based "shape readout" of different nucleic acid conformations.

A modified fluorescent intercalator displacement assay for RNA ligand discovery.

Fluorescent intercalator displacement (FID) is a convenient and practical tool for identifying new nucleic acid-binding ligands. The success of FID is based on the fact that it can be fashioned into a versatile screening assay for assessing the relative binding affinities of compounds to nucleic acids. FID is a tagless approach; the target RNAs and the ligands or small molecules under investigation do not need to be modified in order to be examined. In this study, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. Electrospray ionization mass spectrometry (ESI-MS) results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs (A-site, TAR [transactivation response element], h31 [helix 31], and H69 [helix 69] were identified.

The oxidoreductases LivQ and NeoQ are responsible for the different 6'-modifications in the aminoglycosides lividomycin and neomycin.

AIMS: The 2-deoxystreptamine-containing aminoglycoside antibiotics (AGAs) constitute the largest subgroup of the aminoglycosides. Neomycin (NEO) and lividomycin (LIV) are both representatives of the pseudo-tetrasaccharide group among the NEO-type AGAs. While NEO contains a 6'-NH(2) group, the 6'-position remains unmodified in LIV. The aim of the study was to characterize the substrate specificities of the enzymes involved in the C-6'- and C-6‴-modification in order to explain the different amination patterns. METHODS AND RESULTS: We overproduced and purified the enzymes NeoQ (bifunctional 6'- and 6‴-oxidoreductase) and NeoB (bifunctional 6'- and-6‴-aminotransferase), which had been analysed before (Huang et al. 2007), and compared the enzymatic properties with the corresponding enzymes LivQ (postulated 6‴-oxidoreductase, 72% identity to NeoQ) and LivB (postulated 6‴-aminotransferase, 71% identity to NeoB) from the LIV pathway. By applying a newly established photometric assay, we proved that LivQ oxidized only pseudotetrasaccharidic substrates at the 6‴-position. In contrast, NeoQ accepted also the pseudodisaccharidic paromamine as a substrate and oxidized the 6'- and 6‴-positions on two different precursors of NEO. The aminotransferases LivB and NeoB both transfer NH(2) groups to the 6'-position in the precursor 6'-oxo-paromamine and to the 6‴-position of 6‴-oxo-neomycin C. CONCLUSIONS: The difference in the modification pattern of NEO and LIV at their 6'-positions is based only on the difference in the substrate specificities of the oxidoreductases LivQ and NeoQ, respectively. The aminotransferases LivB and NeoB share identical biochemical properties, and both are capable to transaminate the 6' and also the 6‴-position of the tested AGAs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data provide information to understand the structural variations in aminoglycosides and may be helpful to interpret variations in other natural product bisoynthesis pathways.