Crystal Structure at 1.45 Å Resolution of Alanine Racemase from a Pathogenic Bacterium, Pseudomonas aeruginosa, Contains Both Internal and External Aldimine Forms^,

Abstract

The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands. The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees. This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A. Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures. It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP. Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms. The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.

Pyridoxal 5-Phosphate Enzymes as Targets for Therapeutic Agents

“…AlaR is universal to bacteria, including mycobacteria, and, with a few exceptions [213][214][215][216], is absent in eukaryotes. This taxonomical restriction and the absolute requirement for D-alanine in biosynthesis of prokaryotic cell walls, make AlaR an attractive target for inhibitors that might function as antibiotics [217,218]. Actually, D-alanine is one of the central molecules of the cross-linking step of the assembly of peptidoglycan, which is the backbone of bacterial cell wall.…”

“…1D7K, 2.10 [165] 1F3T, 2.00 (putrescine) [170] 2TOD, 2.00 (eflornithine) [29] 1ORD, 3.00 [163] 1NJJ, 2.45 (G418) [180] 1QU4, 2.90 [29] 7ODC, 1.60 [164] Diaminopimelate decarboxylase (III) 1KNW, 2.10 [209] 1KO0, 2.20 (L-lysine) [209] 1TUF, 2.40 (azalaic acid) [211] 1HKW, 2.80 [210] 1HKV, 2.60 (L-lysine) [210] 1TWI, 2.00 (L-lysine) [211] Alanine racemase (III) 1SFT, 1.90 [13] 1BD0, 1.60 (alanine phosphonate) [222] 1XFC, 1.90 [217] 1EPV, 2.20 (D-cycloserine adduct) [221] 1RCQ, 1.45 [218] 1NIU, 2.20 (L-cycloserine adduct) [221] 1VFH, 2.00 [233] 1VFS, 1.90 (D-cycloserine) [233] 1VFT, 2.30 (L-cycloserine) [233] 2SFP, 1.90 (propionate) [223] Serine hydroxymethyltransferase (I) 1BJ4, 2.65 [250] 1DFO, 2.40 (L-glycine and 5-formyl tetrahydrofolate) [247] 1CJ0, 2.80 [248] 1KKP, 1.93 (L-serine) [249] 1EJI, 2.90 [246] 1KL1, 1.93 (L-glycine) [249] 1KKJ, 1.93 [249] Cystathionine γ-synthase (I) 1CS1, 1.50 [277] 1I41, 3.20 (APPA) [286] 1QGN, 2.90 [278] 1I43, 3.10 (PPCA) [286] 1I48, 3.25 (CTCPO) [286] Cystathionine β-lyase (I) 1CL1, 1.83 [294] 1CL2, 2.20 (L-aminoethoxyvinyl glicine) [284] ( 1IBJ, 2.30 [295] Cystathionine γ-lyase (I) 1N8P, 2.60 [276] Methionine γ-lyase (I) 1GC0, 1.70 …”

Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4

“…The catabolic alanine racemases (e.g. DadX in E. coli) are commonly part of an operon (16), suggesting that the alanine racemase of B. pseudofirmus OF4 might be a catabolic alanine racemase.…”

Structure, Function and Dynamics in the mur Family of Bacterial Cell Wall Ligases

“…In some other Gram-positive bacteria, this interbridge can be formed by residues such as serine, threonine, aspartic acid, lysine or ornithine. 3,4 Structural information is available for the majority of the enzymes involved in peptidoglycan biosynthesis, namely MurA from Escherichia coli 8 and Enterobacter cloacae, 9 MurB from E. coli, 10,11 and S. aureus, 12 E. coli MurG, 13 S. aureus femA 14 and Weissella viridescens femX, 15 along with peripheral enzymes which generate precursors including glutamate racemase (MurI/RacE) from Aquifex pyrophilus 16 and Bacillus subtilis, 17 alanine racemase (alr) from Bacillus stearothermophilus, 18 Pseudomonas aeruginosa, 19 Streptomyces lavendulae 20 and Mycobacterium tuberculosis, 21 and D-Ala-D-Ala ligase (ddl) 22 which produces the dipeptide substrate for MurF. Furthermore, the structures of all four of the amide bond ligases responsible for the addition of the pentapeptide tail to UDPMurNAc are known, and these four enzymes are the subject of the current review.…”