Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase.

Veeraraghavan Usha,Adrian J Lloyd,Guennadi Kozlov,Hasan T Imam,Smita Chauhan,Kalle Gehring,Christopher G Dowson,Claudia A Blindauer,David I Roper,Gurdyal S Besra

doi:10.1038/srep23191

Veeraraghavan Usha, Adrian J Lloyd + Show 8 more

Open Access

https://doi.org/10.1038/srep23191

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Abstract

With the increased incidence of tuberculosis (TB) caused by Mycobacterium tuberculosis there is an urgent need for new and better anti-tubercular drugs. N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a key enzyme in the succinylase pathway for the biosynthesis of meso-diaminopimelic acid (meso-DAP) and L-lysine. DapE is a zinc containing metallohydrolase which hydrolyses N-succinyl L,L diaminopimelic acid (L,L-NSDAP) to L,L-diaminopimelic acid (L,L-DAP) and succinate. M. tuberculosis DapE (MtDapE) was cloned, over-expressed and purified as an N-terminal hexahistidine ((His)6) tagged fusion containing one zinc ion per DapE monomer. We redesigned the DAP synthetic pathway to generate L,L-NSDAP and other L,L-NSDAP derivatives and have characterised MtDapE with these substrates. In contrast to its other Gram negative homologues, the MtDapE was insensitive to inhibition by L-captopril which we show is consistent with novel mycobacterial alterations in the binding site of this drug.

Highlights

The DAP biosynthetic pathway operates only in bacteria and plants
Escherichia coli ArgD (EcArgD) and E. coli DapD (EcDapD) were purified to apparent homogeneity (Supplementary Fig. S1d and S1e) and yields of about 10 mg of EcArgD and 15 mg of EcDapD were obtained per litre of culture
To synthesise MtDapE substrates preparatively, we reconfigured the spectrophotometric assay of L,L-acyl-DAP synthesis as a single pot method using EcArgD, EcDapD and Cgmeso-DAP dehydrogenase (Methods section; Fig. 1a, reaction condition 1)

Summary

Results & Discussion

Overexpression and purification of MtDapE, Cgmeso-DAP dehydrogenase, BaDapF, EcArgD and EcDapD. We tested the possibility that malonyl, butyryl, acetoacetyl, acetyl or propionyl-CoA thiol esters could not support the EcDapD activity efficiently enough to generate detectable quantities of acyl-L,L-DAP analogues over duration of the assay. To synthesise MtDapE substrates preparatively, we reconfigured the spectrophotometric assay of L,L-acyl-DAP synthesis as a single pot method using EcArgD, EcDapD and Cgmeso-DAP dehydrogenase (Methods section; Fig. 1a, reaction condition 1). The assay we developed using our in situ substrate synthesis would be of utility for detection of DapE inhibitors which could potentially have antimicrobial properties Thiols such as L-captopril are potent inhibitors of HiDapE14 and NmDapE10 (Ki values 2.8 μM and 1.8 μM respectively). We developed a cheap and efficient method to access the L,L-NSDAP substrate of Mt-DapE that may support future screening for new DapE-directed antimicrobials

All the plasmids used in this study are listed in Supplementary

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