Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.
Ontology highlight
ABSTRACT:
SUBMITTER:
PROVIDER: S-EPMC3234902 | biostudies-literature | 2011 Dec
REPOSITORIES: biostudies-literature
Similar Datasets
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: Not available
| S-EPMC3023534 | 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: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: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:The transportation of proteins encoded by nuclear genes from plant cytosol to chloroplast is essential for chloroplast functions. Proteins that have a chloroplast transit peptide (cTP) are imported into chloroplasts via translocases on the outer and inner chloroplast envelope. How proteins lacking transit sequence are imported into chloroplast remains largely unknown. During screening of an Arabidopsis population transformed with a hairpin RNA gene-silencing library, we identified some transgenic plants that had active expression of the selectable marker gene, hygromycin phosphotransferase (HPT), but were sensitive to the selection agent, hygromycin B (HyB). Mutant and complementation analysis showed that this HyB sensitivity of transgenic plants was due to silencing of the HS1 (Hygromycin-Sensitive 1) gene. HS1 is localized in the chloroplast and interacts physically with HPT in yeast cells and in planta. Fluorescence and immunoblotting analysis showed that HPT could not be transported effectively into chloroplasts in Aths1, which resulted in Aths1 is sensitivity to hygromycin on higher HyB-containing medium. These data revealed that HS1 is involved in HyB resistance in transgenic Arabidopsis through facilitating cytosol-chloroplast transportation of HPT. Our findings provide novel insights on transportation of chloroplast cTP-less proteins.
| S-EPMC7266939 | biostudies-literature
Project description:The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn(2+)-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosB(Sa)) has been determined to a resolution of 1.15 Å. Cocrystallization of FosB(Sa) with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosB(Sa) contain Zn(2+) in the active site, kinetic analyses of FosB(Sa) indicated that the enzyme is inhibited by Zn(2+) for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.
| S-EPMC3985756 | biostudies-literature
Project description:Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2-containing plasmids or single-copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT-1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild-type C. albicans. We used PCR to fuse CaHygB or SAT-1 to approximately 1 kb of 5' and 3' noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild-type C. albicans strains. Homologous targeting frequencies were approximately 50-70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates.
| S-EPMC4243612 | 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:Hygromycin B is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes. Ribosomal alterations in bacteria conferring resistance to hygromycin B have not been described, prompting us to use a single rRNA allelic derivative of the gram-positive bacterium Mycobacterium smegmatis for investigation of the molecular mechanisms involved in ribosomal resistance to hygromycin B in eubacteria. Resistance mutations were found to localize exclusively in 16S rRNA. The mutations observed, i.e., 16S rRNA U1406C, C1496U, and U1498C (E. coli numbering), are in close proximity to the hygromycin B binding site located in conserved helix 44 of 16S rRNA. The 16S rRNA positions involved in hygromycin B resistance are highly conserved in all three domains of life, explaining the lack of specificity and general toxicity of hygromycin B.
| S-EPMC153343 | biostudies-literature