Abstract
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.
Highlights
Vide essential scaffolds that could be altered with modern medicinal chemical approaches or may be suitable to be reintroduced into the clinic in this current era of pressing need
Structure and Function of aminoglycoside phosphotransferase (APH)[4]-Ia amino sugar rings at the 4 and 6 positions, or the 4 and 5 positions. Hygromycin B (hygB) is unique in that it is comprised of an N-methyl 2-deoxystreptamine ring, linked through the C5-OH to a talose sugar, which in turn forms an orthoester with the unusual amino acid destomic acid (Fig. 1), resulting in a distinctive fused ring structure
Using purified recombinant APH[4]-Ia, we screened a diverse panel of aminoglycoside antibiotics as potential substrates including the 4,6-disubstituted 2-deoxystreptamine-based aminoglycosides, the 4,5-disubstituted 2-deoxystreptamine-based aminoglycosides and the atypical aminoglycosides
Summary
Expression and Purification of APH[4]-Ia—The aph[4]-Ia gene (NCBI accession number V01499) from the retroviral protein expression vector pQCXIH (Clontech) was used as the template for preparation of an overexpressing construct. The gene was amplified with oligonucleotide primers that introduced flanking 5Ј NdeI and 3Ј HindIII sites that were used to clone the gene into vector pET28a to create the expression plasmid pET-APH[4]-Ia. APH[4]-Ia was expressed in E. coli BL21(DE3) (Novagen) and grown overnight in the presence of 50 g/ml kanamycin A in Luria Bertani (LB) broth. The His tag was removed by cleavage with TEV protease overnight at 4 °C in dialysis with the buffer 0.3 M NaCl, 50 mM HEPES, pH 7.5, 5% glycerol, and 0.5 mM tris[2-carboxyethyl]phosphine, followed by binding to nickel-nitrilotriacetic acid resin and capture of flow-through. Crystallography, Data Collection, and Structure Determination—APH[4]-Ia crystals were grown at 22 °C using the hanging drop method, by mixing 1 l of protein at 6.5 mg/ml with 1 l of reservoir solution containing 0.2 M diammonium hydrogen citrate, 16% polyethylene glycol 3350, and 2 mM hygB. The product was recovered with a wash of 0.1% NH4OH and lyophilized
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