Cin, sisomicin, and netilmicin as those in cells producing the wild-type enzyme. The MICs of dibekacin and kanamycin A had been 2- and 4-fold decrease. The MICs of kanamycin A, kanamycin B, and dibekacin created by the APH(2 )-IVa mutant enzyme were identical to those created by E. coli JM83 expressing the wild-type kinase, while the MICs of all other aminoglycosides tested had been 2-fold reduced than those created by the nonmutant enzyme. Steady-state kinetic analysis of the mutant aminoglycoside kinases revealed that the M85Y substitution in APH(two )-IIa resulted in a 10-fold reduce within the Km value for GTP and also a dramatic ( 300-fold) boost within the Km worth for ATP (Table two). On the other hand, the F95Y substitution in APH(2 )IVa resulted in modest decreases in the Km values for both ATP (3-fold) and GTP (4-fold) (Table two). These data indicate that the mutant APH(two )-IIa enzyme lost its ability to make use of ATP as aTABLE 2 NTP substrate profiles for APH(2 )-IIa and APH(two )-IVa and “gatekeeper” mutantskcat (s 1) Enzyme APH(two APH(two APH(two APH(two )-IIa )-IIa M85Y )-IVa )-IVa F95Y ATP 43 two 14 1 0.83 0.02 0.39 0.01 GTP 9.four 0.1 0.84 0.02 0.83 0.01 0.36 0.01 Km ( M) ATP 16 2 5,100 600 one hundred eight 38 four GTP 70 2 7.5 0.five 137 5 36 4 kcat/Km (M ATP (2.7 (2.7 (8.two (1.0 0.4) 0.3) 0.7) 0.1) 106 103 103s 1) GTP (1.3 (1.1 (6.1 (1.0 0.1) 0.1) 0.two) 0.1) 105 105 103aac.asm.orgAntimicrobial Agents and ChemotherapyMutant NTP-Binding TemplatesFIG 1 Stereoviews of the nucleotide-binding pockets in APH(2 )-IIa (A) and APH(two )-IVa (B). The enzymes are shown in ribbon representation (blue). Theavailable binding pocket is shown as a transparent yellow surface. In both cases, the “gatekeeper” residue was excluded in the binding cavity calculation.41102-25-4 Formula An ATP molecule, as bound in APH(2 )-IIa, is shown as a ball-and-stick model at the bottom of every panel and was also excluded in the cavity calculations.1340313-49-6 Data Sheet The secondary binding pocket is indicated by a black asterisk in every panel.PMID:23008002 (A) The APH(two )-IIa “gatekeeper” residue (M85) was mutated to tyrosine (magenta sticks) in silico and is shown in a rotamer conformation similar to that of the original methionine. The residues which line the secondary pocket (V75 and F57), as well as the conserved lysine residue (K42), are shown as cyan sticks. The rotamer conformation of a tyrosine at position 85 directed away in the ATP-binding pocket and into this putative secondary pocket is shown as white sticks. (B) The APH(two )-IVa “gatekeeper” residue (F95) was mutated to tyrosine (magenta sticks) in silico. The secondary pocket, bounded by V78 and V61 (cyan), is able to accommodate each the wild-type phenylalanine (not shown) plus the tyrosine mutant (magenta sticks).cosubstrate, while the cosubstrate specificity from the mutant APH(two )-IVa enzyme did not modify. Antibiotic susceptibility levels made by the APH(two )-IIa M85Y mutant enzyme in E. coli JM83 did not modify for the majority of aminoglycosides tested (Table 1), indicating that the M85Y substitution did not noticeably compromise the stability with the enzyme. The 2-fold lower in MIC values made by the APH(2 )-IVa F95Y mutant was also insignificant and may well have resulted from just a slight lower in the enzyme stability in comparison to that on the parental aminoglycoside phosphotransferase. Based on our kinetic information, the much more pronounced, 4-fold lower in the MIC of kanamycin A made by the APH(two )-IIa M85Y mutant enzyme could have resulted in the transform of its cosubstr.