IMP-43 and IMP-44 Metallo-β-Lactamases with Increased Carbapenemase Activities in Multidrug-Resistant Pseudomonas aeruginosa

Abstract

bTwo novel IMP-type metallo-␤-lactamase variants, IMP-43 and IMP-44, were identified in multidrug-resistant Pseudomonas aeruginosa isolates obtained in medical settings in Japan. Analysis of their predicted amino acid sequences revealed that IMP-43 had an amino acid substitution (Val67Phe) compared with IMP-7 and that IMP-44 had two substitutions (Val67Phe and Phe87Ser) compared with IMP-11. The amino acid residue at position 67 is located at the end of a loop close to the active site, consisting of residues 60 to 66 in IMP-1, and the amino acid residue at position 87 forms a hydrophobic patch close to the active site with other amino acids. An Escherichia coli strain expressing bla IMP-43 was more resistant to doripenem and meropenem but not to imipenem than one expressing bla IMP-7 . An E. coli strain expressing bla IMP-44 was more resistant to doripenem, imipenem and meropenem than one expressing bla IMP-11 . IMP-43 had more efficient catalytic activities against all three carbapenems than IMP-7, indicating that the Val67Phe substitution contributed to increased catalytic activities against carbapenems. IMP-44 had more efficient catalytic activities against all carbapenems tested than IMP-11, as well as increased activities compared with IMP-43, indicating that both the Val67Phe and Phe87Ser substitutions contributed to increased catalytic activities against carbapenems.

Elucidating the Role of Residue 67 in IMP-Type Metallo-β-Lactamase Evolution

“…The V67F mutation (IMP-10) has a significant effect on MICs of carbapenems (8-fold higher for meropenem and doripenem and 4-fold higher for imipenem), which correlates well with the catalytic efficiencies (2-to 4-fold higher for meropenem and doripenem and 1.6-fold higher for imipenem). The V67F mutation in the IMP-type MBLs IMP-43 and IMP-44 has been shown to have impacts on carbapenem susceptibility and activity similar to those of IMP-7 and IMP-11, respectively, which have valine at position 67 (21). Mutation of V67 to alanine or isoleucine also has a positive effect on catalytic efficiencies against meropenem and doripenem but not imipenem.…”

“…This can be extended to IMP-1-V67I, which is almost as efficient as IMP-1, and IMP-1-V67A, which is less efficient, like IMP-10. It appears that penicillins are optimally inactivated when residue 67 is either valine or isoleucine, both of which are branched hydrophobic residues that could pack nicely with the axial methyl groups on the dihydrothiazine ring of penicillins, which has been highlighted in numerous studies (21,25,53). Crystal structures of IMP-1 in complex with a substrate, intermediate, or product are not available; however, in a crystal structure of the MBL NDM-1 in complex with hydrolyzed benzylpenicillin (PDB code 4EYF) (54), the axial methyl group pointing toward loop L3 is very close to V73, the residue that corresponds to position 67 in the BBL numbering scheme (closest COC distance of 4.3 Å in chain A and 4.4 Å in chain B).…”

“…The encoding gene, bla , differs from bla IMP-1 by a point mutation at nucleotide 145 from G to T, which translates into a V67F amino acid change in the substrate binding site. F67 can also be found in the variants IMP-20 (GenBank accession code [GBAC] AB196988), IMP-26 (20), IMP-40 (GBAC AB753457), IMP-41 (GBAC AB753458), IMP-43, and IMP-44 (21). Based on currently published biochemical data, enzymes with F67 show relatively high activity toward cephalosporins and carbapenems (k cat /K m values similar to [19] or up to an order of magnitude higher than [21] those of the corresponding enzymes with V67) but low activity toward penicillins except carbenicillin (k cat /K m values about 1/10 [21] or less [19] of those of the corresponding enzymes with V67).…”

Multidrug-Resistant Sequence Type 235 Pseudomonas aeruginosa Clinical Isolates Producing IMP-26 with Increased Carbapenem-Hydrolyzing Activities in Vietnam

“…S2 in the supplemental material). Compared to IMP-7 and IMP-11, IMP-43 and IMP-44, respectively, showed more-efficient catalytic activities against carbapenems (2). Therefore, the Val67Phe amino acid substitution in IMP-26 may affect its catalytic efficiency against all carbapenems tested, including doripenem, imipenem, meropenem, and panipenem.…”

B1-Metallo-β-Lactamases: Where Do We Stand?

“…The addition of a longer bulky group in Phe compared to Val at position 67 may affect substrate binding resulting in the decreased inactivation of penicillins observed by IMP-10, a single variant of IMP-1 (V67F) [93]. Interestingly, IMP-43 possesses the same amino acid substitution (V67F) compared with IMP-7 (which is close to the IMP-1 cluster), and IMP-44 has two substitutions (V67F and F87S) compared with IMP-11 (which is related to IMP-2) [94]. The V67F substitution in IMP-43 significantly increases the catalytic efficiency for meropenem, whereas the double substitution V67F and F87S in IMP-44 also improves hydrolysis of imipenem and doripenem, compared to IMP-7 and IMP-11, respectively [94].…”