Crystallization and preliminary X-ray crystallographic analysis ofD-phenylglycine aminotransferase fromPseudomonas stutzeriST201

Abstract

d-Phenylglycine aminotransferase (d-PhgAT) catalyzes the reversible transamination of d-phenylglycine to l-glutamate with 2-oxoglutarate as the amino-group acceptor. Crystals of substrate-free Pseudomonas stutzeri d-PhgAT bound to the cofactor pyridoxal-5'-phosphate (PLP) were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 75.155, c = 147.554 A. The asymmetric unit contains one molecule of d-PhgAT and has a solvent content of 50.0%. A complete native X-ray diffraction data set was collected from a single crystal at 100 K to a resolution of 2.3 A.

Effects of Halophilic Peptide Fusion on Solubility, Stability, and Catalytic Performance of D-Phenylglycine Aminotransferase

“…The overexpression and purification of recombinant D-PhgAT were done as previously described [3,11]. Briefly, overnight culture of the recombinant E. coli tuner (DE3) pLysS was used to inoculate 250 ml of Luria Bertani broth (Difco) containing 100 µg/ml of ampicillin (Bio Basic Inc., Canada) and 34 µg/ml of chloramphenicol (Sigma-Aldrich Co. LLC, USA), followed by incubation at 37°C and 200 rpm until OD 600 of 0.6 was reached.…”

“…1), was first reported in 1997 [24]. Crystallography and 3D studies revealed that D-PhgAT is a homodimer enzyme, where each monomer possesses three domains namely a C-terminus, an N-terminus, and a cofactor binding domain with 453 amino acid residues and 47.5 kDa molecular mass [11]. Owing to its stereo-inverting property and the ability to utilize L-glutamic acid as a cheap amino group donor, D-PhgAT is an invaluable catalyst in the single-step synthesis of D-Phg and D-4hydroxyphenylglycine (D-4-OHPhg), which are essential side-chain moieties for manufacturing semisynthetic antibiotics in the penicillin and cephalosporin families.…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…The hypothetical ancestral enzyme might also have had some preference towards the amino group acceptor; in fact, in Fig. 6 most of the tree branches closest to the root represent α-KGspecific ATs (DHNEAT, NEAT, LAT and D-FGAT: for the last enzyme, a PDB-deposited crystal structure exists (2CY8; [87]), which however does not contain bound PLP and lacks other structural details).…”

Use of Enzymes in the Synthesis of Amino Acids

“…once the scope of this aminotransferase is broadened to other classes of D-amino acids by protein engineering -a process which will certainly benefit from the structural information that recently became available [206].…”