Characterization of a New Member of the Flavoprotein Disulfide Reductase Family of Enzymes from Mycobacterium tuberculosis

Abstract

The lpdA (Rv3303c) gene from Mycobacterium tuberculosis encoding a new member of the flavoprotein disulfide reductases was expressed in Escherichia coli, and the recombinant LpdA protein was purified to homogeneity. LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP ؉ per monomer. Although annotated as a probable lipoamide dehydrogenase in M. tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis. The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site. Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone. Significant primary deuterium kinetic isotope effects were observed with [4S-2 H]NADH establishing that the enzyme promotes transfer of the C 4 -proS hydride of NADH. The absence of an isotope effect with [4S-2 H]NADPH, the low K m value of 0.5 M for NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP ؉ (K i ϳ 6 nM) and 2-phospho-ADP-ribose (K i ϳ 800 nM), demonstrate the high affinity of LpdA for 2-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP ؉ rather than NADH/NAD ؉ . Modeling of NADP؉ in the active site revealed that LpdA achieves the high specificity for NADP ؉ through interactions involving the 2-phosphate of NADP ؉ and amino acid residues that are different from those in glutathione reductase. The flavoprotein disulfide reductases (FDRs)1 show high sequence and structural similarities (1, 2). Although most of the FDR enzymes, including lipoamide dehydrogenase (LipDH), glutathione reductase (GR), trypanothione reductase, mycothione reductase, thioredoxin reductase, alkylhydroperoxide reductase, and coenzyme A disulfide reductase catalyze reduction of disulfide-bonded substrates, some do not (1, 2). For example, mercuric ion reductase, NADH peroxidase, NADH oxidase, and 2-ketopropyl-coenzyme M carboxylase/oxidoreductase catalyze reduction of mercuric ion, hydrogen peroxide, molecular oxygen, and the reductive carboxylation of 2-ketopropyl-coenzyme M, respectively. Also, the soluble pyridine nucleotide transhydrogenase (STH) catalyzes the interconversion of the two reduced cellular pyridine nucleotide pools (1, 2).Most of the FDR enzymes are homodimers containing a tightly but noncovalently bound FAD per monomer (1, 2). Reduction of the FAD cofactor by NAD(P)H on the re face of the FAD to generate a transient FADH 2 ⅐NAD(P) ϩ intermediate (3-5) is a common first step in these enzymes. Subsequent electron transfer from FADH 2 to a nonflavin redox center, located on the si face of the FAD, is als...

Lipoic Acid Metabolism in Microbial Pathogens

“…Genomic predictions of three lipoamide dehydrogenase genes in M. tuberculosis are also misleading; among these paralogs, only LpdC, found in the functional PDH complex, is active (7,8). This contrasts with other bacterial species that have multiple E3 paralogs, such as P. aeruginosa, in which each putative E3 gene encodes an active enzyme that functions in specific lipoylated complexes.…”

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

“…The differential expression phenotype of lpdA transcription revealed by our qRT-PCR experiment indicated that the lpdA was probably an early response gene involved in adaption to macrophage environment. The M. tuberculosis LpdA (Rv3303c), despite its significant homology to other LpdA enzymes, does not have lipoamide dehydrogenase activity but instead has transhydrogenase activity and quinone reductase activity that enables transfer of reducing power from the reduced pyridine nucleotide pool to the electron transport chain [58], which could be important for energy production under anaerobic conditions. LpdA is a virulence factor involved in removing reactive oxygen species released by the host cells and thus contributing to in vivo virulence [59].…”

Computational Comparative Study of Tuberculosis Proteomes Using a Model Learned from Signal Peptide Structures

“…The LpdA had been thought as a probable Mtb's dehydrogenases; however, it was verified as a NAD(P)H quinone reductase [49]. The protein of Rv3303c was also supposed to contribute to the virulence because the NAD(P)H quinone reductase may remove reactive oxygene [50].…”

“…The protein structure of O53355 was solved in 2004 [49] and deposited in Protein Data Bank as 1XDI. The N-terminal region forms a part of the structure, and thus it is confirmed that this protein does not have a cleavable signal peptide.…”

“…Since the possible interaction residues R (245), S (213), and Q (214) of the Rv3303c protein do not lie in the signal peptide domain, cleaving signal peptide may not affect the function of quinone reductase [49].…”