Discovery of missing enzyme for tuberculosis iron scavenging pathway

Scientists have discovered that a protein that was originally believed to be involved in tuberculosis antibiotic resistance is actually a "missing enzyme" from the biosynthetic pathway for an agent used by the bacteria to scavenge iron.

The research appears as the "Paper of the Week" in the April 8 issue of the Journal of Biological Chemistry, an American Society for Biochemistry and Molecular Biology journal.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for more morbidity in humans than any other bacteria. The emergence of multi-drug resistant strains of M. tuberculosis has prompted the search for new drug targets and a better understanding of the mechanism of resistance in this bacterium.

Several spans of DNA in the M. tuberculosis genome have been annotated as antibiotic resistance genes due to their sequence similarity to existing antibiotic resistance genes. Dr. Edward N. Baker of the University of Auckland in New Zealand explains, "Generally the sequence of the open reading frame is compared with the sequences of genes for other proteins (most of which are from different species) in sequence databases. If a close match is found, it is assumed that the function is the same or similar."

Rv1347c is one of these annotated antibiotic resistance genes in M. tuberculosis. It encodes a putative aminoglycoside N-acetyltransferase that is thought to be involved in resistance to aminoglycoside antibiotics such as streptomycin.

"The aminoglycoside antibiotics have sugar rings with amino groups attached," explains Dr. Baker. "The N-acetyltransferase chemically modifies the sugar amino group by transferring an acetyl group to it. This inactivates the antibiotic because it can no longer fit into its target."

However, in vitro biochemical assays have failed to demonstrate aminoglycoside N-acetyltransferase activity in Rv1347c. By solving the three-dimensional structure of Rv1347c, Dr. Baker and his colleagues have discovered that the enzyme most likely plays an entirely different role in M. tuberculosis.

"What the structure showed, when combined with careful analysis of the sequence, its neighbors in the genome, and the fact that its gene was also regulated by iron, was that Rv1347c was almost certainly a "missing enzyme" from the pathway for biosynthesis of the iron scavenging agent mycobactin," recalls Dr. Baker.

"Mycobactin is a small molecule which binds iron very tightly. Bacteria synthesize it so that they can acquire the iron they need to grow – it is secreted out into the external environment where it scavenges iron and then (with iron bound to it) it is taken up by the bacterium again."

Although Rv1347c is not involved in antibiotic resistance, it still remains a target for the design of new anti-TB drugs. "Enzymes that synthesize mycobactin are drug targets, because if mycobactin biosynthesis is stopped, the bacterium cannot acquire the iron that it needs for survival," explains Dr. Baker. "Importantly this seems to be true even of the bacteria that are taken up by macrophages in the lung and enter a dormant state – these are the hardest to attack with drugs."