Enzymes involved in thiamin biosynThesis and crystal structure of AhpE: 1-Cys peroxiredoxin from M. tuberculosis

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is still the biggest killer among the infectious diseases, killing about 2-3 million people around the world each year. Although TB morbidity and mortality rates steadily dropped during the 20th century, the rise in TB incidence in the last two decades, partly due to the combination of TB and HIV and the emergence of multidrug-resistant strains of the bacteria, has caused major concern. M. tuberculosis is characterized by its ability to persist in vivo and become dormant for years, only to reactivate when the host immune response is compromised, limiting the actions of current drugs. Novel and more effective anti-tuberculosis drugs are needed, and with the completion of the sequence and annotation of the M. tuberculosis genome, the development of efficient mutagenesis techniques and strategies, and structure-based drug design, will make their development possible. Thiamine-dependent enzymes, such as pyruvate dehydrogenase, play a particularly important role in carbohydrate metabolism. M. tuberculosis has no homologue of i/zz'K, which is required for the salvage of thiamin, thus, the thiamin biosynthesis pathway is likely to be particularly important for the survival of M. tuberculosis inside infected macrophages. Moreover, the thiamin biosynthesis pathway is only present in plants and microorganisms, not humans, which implies that inhibitors designed for the thiamin biosynthesis pathway might not affect the human cell, and that these enzymes may therefore be an ideal target for drug design. Six essential proteins from M. tuberculosis (ThiO, ThiG, ThiC, ThiD, ThiE, and ThiL) involved in thiamin biosynthesis have been cloned and expressed, but ThiO (Rv0415), ThiG (Rv0417), and ThiD (Rv0422c) were insoluble, and thus not worked on any further. ThiC (Rv0423c), ThiE (Rv0414c), and ThiL (Rv2977c) were partially soluble. ThiC (Rv0423c), for which no homologous structure is available so far, catalyzes the atomic rearrangement of aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethyl-2-methylpyrimidine monophosphate (HMP-P), and has a stable trypsin protease resistant core structure. Some tiny needle crystals of ThiC were obtained, but the crystals disappeared after two weeks, which is possibly related to the degradation of the protein. ThiE (Rv0441c) was crystallized into thin plate crystals, which showed anisotropic diffraction to 2.7 A resolution. ThiL (Rv2977c) crystallized into single crystals by microseeding technique. However, the crystals did not grow large enough for data collection. All living systems require protection against the damaging effects of reactive oxygen species. The genome of Mycobacterium tuberculosis encodes a number of peroxidases that are thought to be active against organic and inorganic peroxides, and are likely to play a key role in the ability of the organism to survive within the phagosomes of macrophages. The open reading frame Rv2238c in M. tuberculosis encodes a 153-residue protein AhpE, which is a peroxidase of the 1-Cys peroxiredoxin (Prx) family. Two crystal structures of AhpE have been determined from native crystals and sodium bromide soaked crystals. In the native structure, the active site peroxidatic cysteine Cys45 is in its oxidized, sulfenic acid (S-O-H) state. A second crystal form of AhpE, obtained after soaking in sodium bromide, reveals the reduced form of Cys45. In addition to the different states of Cys45, the conformational changes in an external p9-a5 loop allow Argll6, an important highly conserved residue responsible for enhancing the reactivity of Cys45, to move away from or towards Cys45 SY. These conformational changes also open up or close a channel leading to the active site. This long hydrophobic channel is identified as the likely binding site for a physiological oxidant and the conformational change is inferred to be important for the reaction cycle of AhpE. AhpE forms a tightly-associated dimer and a ring-like octamer. Although the significance of the octamer found for AhpE is not known, it parallels the decamers found for other Prxs. The unwinding of the end of the first helix in the oxidized form may be favorable for the regeneration of the oxidized peroxidatic cysteine, suggesting that the AhpE adopts the same regeneration mechanism as that of the AhpC from Salmonella typhimurium. The functional studies confirm that the two conformation states of Argl 16 exist in an equilibrium in solution.