Mycobacterium tuberculosis appears to lack α‐ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes

Abstract

SummaryMycobacterium tuberculosis (Mtb) persists for prolonged periods in macrophages, where it must adapt to metabolic limitations and oxidative/nitrosative stress. However, little is known about Mtb's intermediary metabolism or antioxidant defences. We recently identified a peroxynitrite reductase-peroxidase complex in Mtb that included products of the genes sucB and lpd , which are annotated to encode the dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3) components of a a a a -ketoglutarate dehydrogenase (KDH). However, we could detect no KDH activity in Mtb lysates, nor could we reconstitute KDH by combining the recombinant proteins SucA (annotated as the E1 component of KDH), SucB and Lpd. We therefore renamed the sucB product dihydrolipoamide acyltransferase (DlaT). Mtb lysates contained pyruvate dehydrogenase (PDH) activity, which was lost when the dlaT gene (formerly, sucB ) was disrupted. Purification of PDH from Mtb yielded AceE, annotated as an E1 component of PDH, along with DlaT and Lpd. Moreover, anti-DlaT antibody coimmunoprecipitated AceE. Finally, recombinant AceE, DlaT and Lpd, although encoded by genes that are widely separated on the chromosome, reconstituted PDH in vitro with K m values typical of bacterial PDH complexes. In sum, Mtb appears to lack KDH. Instead, DlaT and Lpd join with AceE to constitute PDH.

Variant tricarboxylic acid cycle in Mycobacterium tuberculosis : Identification of α-ketoglutarate decarboxylase

“…Mtb lysates were prepared as described (16). E. coli, Mycobacterium smegmatis, and Mycobacterium bovis bacillus Calmette-Guérin were grown in LB or 7H9 medium at 37°C.…”

“…The Rv1248c protein was purified from recombinant E. coli as described (16). MtbH37Rv gabD1 (Rv0234c) and gabD2 (Rv1731) genes were amplified from genomic DNA by PCR with Pfu DNA polymerase using primers containing 5Ј NdeI and 3Ј NheI sites.…”

“…Ferricyanide reductase. Ferricyanide reductase was assayed as described (16). Linked assay of Rv1248c protein and GabD1 or GabD2.…”

“…We began by testing Rv1248c for reactions with ␣-ketoglutarate other than its dehydrogenation. Recombinant Rv1248c protein was purified to homogeneity as described (16). Immunoblot with antiserum raised against the pure protein documented expression of Kgd in Mtb during both logarithmic growth and stationary phase (Fig.…”

“…Our previous studies (16) suggest that DlaT and Lpd do not conform to their annotations (http:͞͞genolist.pasteur.fr͞ TubercuList) as components of KDH. In fact, Mtb appears to lack KDH, and DlaT and Lpd serve as components of PDH instead.…”