Biochemical and structural studies of the Mycobacterium tuberculosis O6-methylguanine methyltransferase and mutated variants.

Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the host's infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O(6)-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O(6)-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G · C-to-A · T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology.

Human kynurenine aminotransferase II--reactivity with substrates and inhibitors.

Kynurenine aminotransferase (KAT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of kynurenine, an intermediate of the tryptophan degradation pathway, into kynurenic acid, an endogenous antagonist of ionotropic excitatory amino acid receptors in the central nervous system. KATII is the prevalent isoform in mammalian brain and a drug target for the treatment of schizophrenia. We have carried out a spectroscopic and functional characterization of both the human wild-type KATII and a variant carrying the active site mutation Tyr142→Phe. The transamination and the ß-lytic activity of KATII towards the substrates kynurenine and α-aminoadipate, the substrate analog ß-chloroalanine and the inhibitors (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate were investigated with both conventional assays and a novel continuous spectrophotometric assay. Furthermore, for high-throughput KATII inhibitor screenings, an endpoint assay suitable for 96-well plates was also developed and tested. The availability of these assays and spectroscopic analyses demonstrated that (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are poor substrates that undergo slow transamination.

Biochemical and structural investigations on kynurenine aminotransferase II: an example of conformation-driven species-specific inhibition?

Kynurenic acid (KYNA), one of the metabolites belonging to the kynurenine pathway, has been described as an important neuroprotective compound, its unbalancing being associated with several pathological conditions. In human brain, the majority of KYNA production is sustained by kynurenine aminotransferase II (KAT II). A selective KAT II inhibitor would be an important pharmacological tool, since it would reduce KYNA formation without causing complete depletion of this neuroprotector. (S)-(4)-(ethylsulfonyl)benzoylalanine (S-ESBA), described as a potent and selective inhibitor of rat KAT II, is unfortunately ineffective towards the human enzyme although the two orthologs share a remarkably high degree of sequence identity. We investigated the molecular basis for this intriguing species-specificity by adopting a site-directed mutagenesis and structural approach. We propose that the source of the inhibitor specificity toward the rat enzyme could reside on S-ESBA interaction/interference with a flexible loop that controls ligand admission to the active site by a classical induced-fit mechanism. Our data further highlights that even in case of highly conserved molecular targets, the flexibility of catalytically important structural elements can have a significant impact on the selectivity of inhibitor action.