Aminoglycoside-Modifying Enzymes: Catalytic Mechanisms and Properties

Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes transfer the phosphoryl group of ATP to the 3'-hydroxyl of the antibiotics. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa were carried out with kanamycin A, neamine, their respective difluorinated analogues and D198A mutant APH(3')-Ia, in conjunction with measurements of thio effects and solvent viscosity effects to shed light on the details of the turnover chemistry. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with non-fluorinated substrates. Residue 198 is the proposed general base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophile participation in the transition state for the phosphate transfer reaction. Pre-steady-state kinetics demonstrated that APH(3')-Ia and APH(3')-IIa in both ternary and binary complexes facilitate an ATP hydrolase activity ('ATPase'). Since these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. ATPase activity would potentially consume as much as several fold of the total existing ATP. Studies with bacteria harboring the aph(3')-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3')-Ia gene, such that approximately 50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.A newly discovered bifunctional antibiotic resistance enzyme (ANT(3')-Ii/AAC(6')-IId) from Serratia marcescens catalyzes adenylation of streptomycin and spectinomycin on the 3'- and 9-hydroxyl groups, respectively, and acetylation of kanamycin A on the 6'-amine. The adenyltransferase domain appears to be specific to spectinomycin and streptomycin, while the acetyltransferase domain shows a broad substrate profile. Studies using initial velocity patterns, dead-end and product inhibitions, solvent isotope effects, and solvent viscosity effects reveal that adenyltransferase domain catalyzes the reaction by a Theorell-Chance kinetic mechanism, where ATP binds to the enzyme prior to the aminoglycoside and the modified antibiotic is the last product to be released. The acetyltransferase domain follows an ordered Bi-Bi kinetic mechanism, in which the antibiotic is the first substrate that binds to the active site and coenzyme A is released prior to the modified aminoglycoside. Another bifunctional aminoglycoside-modifying enzyme (AAC(3)-Ib/AAC(6')-Ib') from Pseudomonas aeruginosa catalyzes acetylation of aminoglycoside antibiotics. AAC(3)-Ib is specific to gentamicin and fortimicin A among a dozen of aminoglycosides. Initial velocity and inhibition patterns and the solvent isotope effects indicate that AAC(3)-Ib catalyzes its reaction through an ordered Bi-Bi kinetic mechanism where acetyl-CoA binds first to the enzyme followed by aminoglycoside and the acetylated aminoglycoside is released from the active site prior to coenzyme A. The solvent viscosity effects suggest that the product release is a rate-limiting step in catalysis.