Isolation of a 43 kDa protein from Mycobacterium tuberculosis H₃₇Rv and its identification as a pyridine nucleotide transhydrogenase

Abstract

A 43 kDa protein (TB43) was isolated from the cell sonicate (CS) of Mycobacterium tuberculosis H37Rv with immobilized metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid column. Two-dimensional electrophoresis of the IMAC fraction showed a major spot with an M(r) of 43,000 and a pI of approximately 6.0. The N-terminal amino acid sequence of TB43 was met-arg-val-gly-ile-pro-asn-glu-thr-lys-asn-asn-glu-phe-arg-val-ala- ile-thr-pro-ala. It showed 86% homology with the N-terminal end of the alanine dehydrogenase of Myco. tuberculosis and 65% homology with the N-terminal end of the alpha-subunit of the Escherichia coli pyridine nucleotide transhydrogenase (Tsh). TB43 did not show any alanine dehydrogenase activity and did not react with monoclonal antibody (MAb) HBT10, which is known to recognize the 40 kDa alanine dehydrogenase of Myco. tuberculosis. It was also not recognized by MAb F29-29 which is known to react with a 43 kDa protein of Myco. tuberculosis complex. This protein exhibited strong Tsh activity. A similar 43 kDa protein showing Tsh activity was also isolated by IMAC from Myco. bovis CS. However, the pI of the protein was approximately 7.0. A similar protein could not be isolated from the CS or culture filtrate of Myco. bovis BCG and Myco. tuberculosis H37Ra. TB43 is a cell-associated pyridine nucleotide transhydrogenase and is distinct from the 40/44 kDa secreted alanine dehydrogenase of Myco. tuberculosis.

Morpholine-induced formation of l -alanine dehydrogenase activity in Mycobacterium strain HE5

“…2). A second enzyme, designated as TB43, has been isolated from M. tuberculosis and shows a high sequence similarity to various L-alanine dehydrogenases (Deshpande et al 1994). This 43-kDa protein, which has no alanine dehydrogenase activity, was identified as pyridine nucleotide transhydrogenase.…”

“…Andersen et al (1992), 3Kuroda et al (1990), 4 Jaacks Siranosian et al(1993), 5Kuroda et al (1990), 6 Sawa et al (1994, and 7Deshpande et al (1994). Amino acids that are identical in all sequences are indicated by bold letters…”

Immune recognition surface construction of Mycobacterium tuberculosis epitope-specific antibody responses in tuberculosis patients identified by peptide microarrays

“…40 This is the first study to present thioredoxin C (TrxC) as an immunologically relevant M. tuberculosis target, although its involvement in mycobacterial detoxification mechanisms has been reported previously. [41][42][43]47 This likewise applies to TB43, whose role in M. tuberculosis cell wall maintenance is already known, 44,45 and rpoB, which has long been used as the genetic marker for M. tuberculosis resistance to rifampicin. [46][47][48] Further studies are required for validation of these proteins as legitimate biomarkers.…”

Identification of Lysine 74 in the Pyruvate Binding Site of Alanine Dehydrogenase from Bacillus subtilis

“…Surprisingly, no other amino acid dehydrogenase or hydroxyacid dehydrogenase was found to be significantly similar to alanine dehydrogenase, but the enzyme was found to be similar to the N-terminal sequence of pyridine nucleotide transhydrogenase, suggesting a similar folding of these two protein segments (15). However, no alanine dehydrogenase activity was detected in M. tuberculosis pyridine nucleotide transhydrogenase (56), suggesting that even if they have a similar structure, their active site is different. In agreement with this active-site difference, pyridine nucleotide transhydrogenase also lacks lysine 74, which was found to be essential for the activity of alanine dehydrogenase (Table III).…”