ABCMdb

ABCC8 p.Ser405Asn

Predicted by SNAP2:	A: N (53%), C: D (75%), D: D (71%), E: D (85%), F: D (71%), G: N (53%), H: D (80%), I: D (66%), K: D (59%), L: D (71%), M: D (80%), N: N (66%), P: D (75%), Q: D (59%), R: D (71%), T: N (87%), V: D (66%), W: D (75%), Y: D (66%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: D, K: N, L: D, M: N, N: N, P: D, Q: N, R: N, T: N, V: D, W: N, Y: N,

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Publications
[hide] Inactivation kinetics of a new target of beta-lact... J Biol Chem. 2011 Jul 1;286(26):22777-84. Epub 2011 May 4. Triboulet S, Arthur M, Mainardi JL, Veckerle C, Dubee V, Nguekam-Moumi A, Gutmann L, Rice LB, Hugonnet JE
Inactivation kinetics of a new target of beta-lactam antibiotics.
J Biol Chem. 2011 Jul 1;286(26):22777-84. Epub 2011 May 4., [PMID:21543331]

Abstract [show]
Peptidoglycan is predominantly cross-linked by serine DD-transpeptidases in most bacterial species. The enzymes are the essential targets of beta-lactam antibiotics. However, unrelated cysteine LD-transpeptidases have been recently recognized as a predominant mode of peptidoglycan cross-linking in Mycobacterium tuberculosis and as a bypass mechanism conferring resistance to all beta-lactams, except carbapenems such as imipenem, in Enterococcus faecium. Investigation of the mechanism of inhibition of this new beta-lactam target showed that acylation of the E. faecium enzyme (Ldt(fm)) by imipenem is irreversible. Using fluorescence kinetics, an original approach was developed to independently determine the catalytic constants for imipenem binding (k(1) = 0.061 muM(-1) min(-1)) and acylation (k(inact) = 4.5 min(-1)). The binding step was limiting at the minimal drug concentration required for bacterial growth inhibition. The Michaelis complex was committed to acylation because its dissociation was negligible. The emergence of imipenem resistance involved substitutions in Ldt(fm) that reduced the rate of formation of the non-covalent complex but only marginally affected the efficiency of the acylation step. The methods described in this study will facilitate development of new carbapenems active on extensively resistant M. tuberculosis.

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No. Sentence Comment
61 Derivatives of pET2818 containing the same portion of the ldtfm gene with mutations leading to G430S, S405N, or both amino acid substitutions were constructed as described in the supplemental "Experimental Procedures". Login to comment
152 Identification of Amino Acid Substitutions in Ldtfm-Sequencing the ldtfm gene of mutant S11 revealed two point mutations that led to amino acid substitutions S405N and G430S, both located in the catalytic domain of the LD-transpeptidase (supplemental Fig. S3). Login to comment
153 Sequencing of ldtfm from the intermediary mutants showed that the two mutations appeared sequentially at the eighth (S405N) and 11th (G430S) selection steps. Login to comment
154 Substitution S405N is located at the entrance of the catalytic cavity in the loop connecting sheets beta13 to beta14. Login to comment
157 Kinetics of Inactivation of LD-Transpeptidases from Mutants Resistant to Imipenem-In order to analyze the impact of substitutions G430S and S405N on the catalytic constants, we constructed and purified derivatives of Ldtfm harboring these two substitutions alone or in combination. Login to comment
174 The second substitution, S405N alone, caused moderate decreases in kinact (from 4.5 to 1.6 min-1 ) and k1 (from 0.065 to 0.030 ␮M -1 min-1 ). Login to comment
176 Impact of amino acid substitutions S405N and G430S on the kinetics of Ldtfm inactivation by imipenem. Login to comment
223 A decrease in the efficiency of enzyme acylation was expected to result in increased resistance to imipenem, and this possibility was explored by selecting mutant S11 and analyzing the impact of substitutions S405N and G430S on the kinetics of Ldtfm inactivation (Fig. 7). Login to comment