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Biochemical characterisation of IMP-16, a novel IMP variant harboured in a class I integron of a Pseudomonas aeruginosa clinical isolate from Brazil

Abstract number: 902_p699

Mendes R.E.

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Objectives:

Several variants of IMP-type enzymes have been identified and they are from 1 to 17% amino acid residues divergent from IMP-1. In some cases, they also present with different kinetic properties. Here we report the biochemical characterisation of a new IMP variant, IMP-16, that was found in Pseudomonas aeruginosa index strain (101-4704) clinical isolate from Brasilia (Brazil).

Methods:

Primers targeting the 5’CS and 3’CS regions of class 1 integron were used to amplify the blaIMP-16 containing integron. These primers yielded PCR products, which were sequenced on both strands using DuPont Automated systems. After integron sequence analysis, the blaIMP-16 gene was subcloned into expression vector pPCRScriptCam SK+ and overexpressed in Escherichia coli DH5alfa. The IMP-16 protein was purified by Fast Performance Liquid Chromatography. Kinetic properties were determined with several beta-lactam substrates measuring hydrolytic activity under initial rate conditions.

Results:

The N-terminus of the IMP-16 showed typical features of bacterial signal peptides that target proteins to the periplasmic space and the most likely cleavage site is located between the alanine at positions 18 and glycine at position 19. This produces a mature protein of 25 266 Da with a theoretical pI of 6.5. IMP-16 is a new IMP variant that differs from IMP-1 by 15% amino acid residues, being one of the most divergent variants so far identified. It is more similar to IMP-8 and IMP-11 (89.8 and 90.3%, respectively) at the level of mature protein. E. coli DH5alfa harbouring the blaIMP-16 recombinant plasmid showed a similar beta-lactam resistance profile when compared with the index strain (101-4704), being resistant to nearly all beta-lactam tested, apart from aztreonam and carbapenems. The IMP-16 enzyme was overproduced in E. coli and purified (>95%). Preliminary kinetic analysis revealed Km values of 38, 98, 0.05 and 10 mm, and Kcat values of 0.05, 0.24, 2.0 and 1.2 /s for meropenem, imipenem, penicillin and nitrocefin, respectively.

Conclusions:

IMP-16 is a new highly divergent IMP variant detected in P. aeruginosa. Kinetic parameters showed the great structural and functional plasticity among this group of clinical important enzymes.

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Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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