The 1.9 Å Crystal Structure of Alanine Racemase from Mycobacterium tuberculosis Contains a Conserved Entryway into the Active Site^,

Abstract

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.

Structure of the Mycobacterium tuberculosis d -Alanine: d -Alanine Ligase, a Target of the Antituberculosis Drug d -Cycloserine

“…The D-alanine racemase and Ddl genes have both been identified as essential genes in M. tuberculosis by two methods of genome-wide analysis based on transposon mutagenesis at either saturation or subsaturation levels (22,36). Indeed, the lethal effect of DCS on M. tuberculosis may be due to the inhibition of both Ddl and D-alanine racemase, whose crystal structure has been solved (24) Association/dissociation equilibrium constants have not been reported for Ddl of any species; however, many K m values have (47). Our binding data are consistent with previous kinetic studies from other species which show both high-affinity and low-affinity events.…”

Protein targets for structure-based anti-Mycobacterium tuberculosis drug discovery

“…1). These knowledge of detailed structural investigation and related biochemical analysis made this enzyme a challenging but important target for drug design against TB; furthermore, the related studies provided valuable insights about the precise mechanism of D-cycloserine inhibition against drug-resistant TB (Feng and Barletta, 2003;LeMagueres et al, 2005).…”

The Structure of Serine Palmitoyltransferase; Gateway to Sphingolipid Biosynthesis

“…This phenomenon was also observed in the structure of the alanine racemase from Mycobacterium tuberculosis. 41 It is known that large conformational changes occur during the catalytic cycle of the enzymes from the AOS family thus enabling them to control the bond breaking and forming steps. 11,12,15 Structural impact of human HSAN1 mutations A number of human diseases have been discovered that are caused by defects in sphingolipid catabolism.…”