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Overexpression of a newly identified d‐amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion

2017; Wiley; Volume: 107; Issue: 2 Linguagem: Inglês

10.1111/mmi.13877

1365-2958

Roman Mortuza, Htin Aung, George Taiaroa, Helen K. Opel‐Reading, Torsten Kleffmann, Gregory M. Cook, Kurt L. Krause,

Bacterial Genetics and Biotechnology

Summary Glutamate racemase (MurI) has been proposed as a target for anti‐tuberculosis drug development based on the inability of Δ murI mutants of Mycobacterium smegmatis to grow in the absence of d‐glutamate. In this communication, we identify Δ murI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these Δ murI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT‐qPCR and transcriptional fusion analyses revealed that the Δ murI suppressor mutant overexpressed MSMEG_5795 14‐fold compared to the isogenic wild‐type. MSMEG_5795, which is annotated as 4‐amino‐4‐deoxychorismate lyase (ADCL) but which also has homology to d‐amino acid transaminase (d‐AAT), was expressed, purified and found to have d‐AAT activity and to be capable of producing d‐glutamate from d‐alanine. Consistent with its d‐amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both Δ murI deletion mutants and alanine racemase (Δ alr ) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis .