New Tricks for an Old Antibiotic♦ : Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme
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PROVIDER: S-EPMC3023534 | biostudies-literature | 2011 Jan
REPOSITORIES: biostudies-literature
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| S-EPMC3234902 | biostudies-literature
Project description:The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.
| S-EPMC3023493 | biostudies-literature
Project description:The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.
| S-EPMC4051501 | biostudies-literature
Project description:The APH(2?)-Ia aminoglycoside resistance enzyme forms the C-terminal domain of the bifunctional AAC(6')-Ie/APH(2?)-Ia enzyme and confers high-level resistance to natural 4,6-disubstituted aminoglycosides. In addition, reports have suggested that the enzyme can phosphorylate 4,5-disubstituted compounds and aminoglycosides with substitutions at the N1 position. Previously determined structures of the enzyme with bound aminoglycosides have not indicated how these noncanonical substrates may bind and be modified by the enzyme. We carried out crystallographic studies to directly observe the interactions of these compounds with the aminoglycoside binding site and to probe the means by which these noncanonical substrates interact with the enzyme. We find that APH(2?)-Ia maintains a preferred mode of binding aminoglycosides by using the conserved neamine rings when possible, with flexibility that allows it to accommodate additional rings. However, if this binding mode is made impossible because of additional substitutions to the standard 4,5- or 4,6-disubstituted aminoglycoside architecture, as in lividomycin A or the N1-substituted aminoglycosides, it is still possible for these aminoglycosides to bind to the antibiotic binding site by using alternate binding modes, which explains the low rates of noncanonical phosphorylation activities seen in enzyme assays. Furthermore, structural studies of a clinically observed arbekacin-resistant mutant of APH(2?)-Ia revealed an altered aminoglycoside binding site that can stabilize an alternative binding mode for N1-substituted aminoglycosides. This mutation may alter and expand the aminoglycoside resistance spectrum of the wild-type enzyme in response to newly developed aminoglycosides.
| S-EPMC6021647 | biostudies-literature
Project description:Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6')-Ie-APH(2'')-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6')-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2'')-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2'')-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6')-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6')-Ie enzyme is joined to APH(2'')-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2'')-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6')-Ie-APH(2'')-Ia enzyme.
| S-EPMC4188014 | biostudies-literature
Project description:Despite the availability of Mycobacterium tuberculosis (Mtb) drugs for over 50 years, tuberculosis (TB) remains at pandemic levels. New drugs are urgently needed for resistant strains, shortening duration of treatment, and targeting different stages of the disease, especially for treatment during human immunodeficiency virus co-infection. One solution to the conundrum that antibiotics kill the bacillus yet select for resistance is to target the host rather than the pathogen. Here, we discuss recent progress in so-called 'host-directed therapeutics' (HDTs), focusing on two general mechanistic strategies: (i) HDTs that disrupt Mtb pathogenesis in macrophages and (ii) immunomodulatory HDTs that facilitate protective immune responses that kill Mtb or reduce deleterious responses that exacerbate disease. HDTs hold significant promise as adjunctive therapies in that they are less likely to engender resistance, will likely have efficacy against antibiotic-resistant strains, and may have activity against non-replicating Mtb. However, TB is a complex and variegated disease, and human populations exhibit significant diversity in their immune responses to it, which presents a complicated landscape for HDTs to navigate. Nevertheless, we suggest that a detailed mechanistic understanding of drug action, together with careful selection of disease stage targets and dosing strategies may overcome such limitations and allow the development of HDTs as effective adjunctive treatment options for TB.
| S-EPMC4571192 | biostudies-literature
Project description:Aminoglycoside-2"-O-nucleotidyltransferase ANT(2")-Ia is an aminoglycoside resistance enzyme prevalent among Gram-negative bacteria, and is one of the most common determinants of enzyme-dependant aminoglycoside-resistance. The following report outlines the use of our recently described oxidopyrylium cycloaddition/ring-opening strategy in the synthesis and profiling of a library of synthetic α-hydroxytropolones against ANT(2")-Ia. In addition, we show that two of these synthetic constructs are capable of rescuing gentamicin activity against ANT-(2")-Ia-expressing bacteria.
| S-EPMC4798002 | biostudies-literature
Project description:Aminoglycoside (6') acetyltransferase-Ie/aminoglycoside (2?) phosphotransferase-Ia [AAC(6')-Ie/APH(2?)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5'-[?,?-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (R(G)) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage.
| S-EPMC3318351 | biostudies-literature
Project description:Understanding the mechanism by which streptomycin binds to the small subunit of the mitoribosome may help researchers design less toxic derivatives of this antibiotic.
| S-EPMC9731568 | biostudies-literature
Project description:Achromobacter denitrificans is an environmental opportunistic pathogen that is infecting a large number of immunocompromised patients. A more recently identified strain from the historical collection of strains of Achromobacter denitrificans is Achromobacter mucicolens. In hosts with a variety of underlying diseases, Achromobacter spp. can induce a wide spectrum of disorders. Because of the bacterium's intrinsic genetic constitution and resistance gained over time, antibiotics are challenged to handle A. mucicolens. Due to the fact that A. mucicolens is rare and its taxonomy is not completely understood, it is difficult to define clinical symptoms, acquisition risk factors, and thus the best therapeutic course of action. To help comprehend this intrinsic and acquired resistance, we analyzed the entire genome of the A. mucicolens IA strain and utilized bioinformatics methods to estimate the strain's probable drug resistance profile. In our study, we have isolated and cultured a clinically important A. mucicolens strain and subjected it to antimicrobial susceptibility tests against antibiotics in the Vitek 2 testing system. The strain's genome sequence as well as an investigation of 27 of its phenotypic traits provides important information regarding this pathogen. The genome of this A. mucicolens IA strain possesses a number of antibiotic resistance genes that code for efflux pump systems and other antibiotic-regulating as well as -modifying enzymes. Our research analysis predicted genes involved in drug resistance, including genes for efflux pump systems, antibiotic efflux, antibiotic inactivation, and antibiotic target alteration. In vitro studies validated the genomic evidence for its ability to exhibit resistance against a wide range of antibiotics. Our investigation paves the way for more research on understanding the functioning of the key discovered genes that contribute toward the pathogenicity of A. mucicolens and hence gives new information and treatment options for this emerging pathogen. IMPORTANCE Achromobacter species are well-known opportunistic human pathogens that can be found in water and soil and most commonly in hospital settings. They thrive in immunocompromised individuals, producing sporadic cases of pneumonia, septicemia, peritonitis, urinary tract infections, and other illnesses. Achromobacter strains are inherently resistant to a wide spectrum of antibiotics, making them difficult to treat promptly. The strain under study, A. mucicolens, was notably resistant to various antibiotics, and the infection could be controlled only after several rounds of prescription medications at different doses. This consumed a lot of time and put the already immunosuppressed leukemic patient through a great ordeal. The study aimed to raise awareness about the importance of the Achromobacter bacterium's lethality, and doctors should evaluate the bacterium's potential for resistance before prescribing antibiotics. Sanitation and other precautions should also be implemented in hospitals and other public places.
| S-EPMC9045304 | biostudies-literature