Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein.

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 A resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop of one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the gamma-phosphate of the ATP molecule replacing the Mg(2+) position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3(10) helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.

Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors.

S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 A resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.

High resolution crystal structures of Mycobacterium tuberculosis adenosine kinase: insights into the mechanism and specificity of this novel prokaryotic enzyme.

Adenosine kinase (ADK) catalyzes the phosphorylation of adenosine (Ado) to adenosine monophosphate (AMP). It is part of the purine salvage pathway that has been identified only in eukaryotes, with the single exception of Mycobacterium spp. Whereas it is not clear if Mycobacterium tuberculosis (Mtb) ADK is essential, it has been shown that the enzyme can selectively phosphorylate nucleoside analogs to produce products toxic to the cell. We have determined the crystal structure of Mtb ADK unliganded as well as ligand (Ado) bound at 1.5- and 1.9-A resolution, respectively. The structure of the binary complexes with the inhibitor 2-fluoroadenosine (F-Ado) bound and with the adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (non-hydrolyzable ATP analog) bound were also solved at 1.9-A resolution. These four structures indicate that Mtb ADK is a dimer formed by an extended beta sheet. The active site of the unliganded ADK is in an open conformation, and upon Ado binding a lid domain of the protein undergoes a large conformation change to close the active site. In the closed conformation, the lid forms direct interactions with the substrate and residues of the active site. Interestingly, AMP-PCP binding alone was not sufficient to produce the closed state of the enzyme. The binding mode of F-Ado was characterized to illustrate the role of additional non-bonding interactions in Mtb ADK compared with human ADK.