Z-D-phenylglycine

Background: D-Phenylglycine aminotransferase (D-PhgAT) is highly beneficial in pharmaceutical biotechnology. Like many other enzymes, D-PhgAT suffers from low stability under harsh processing conditions, poor solubility of substrate, products and occasional microbial contamination. Incorporation of miscible organic solvents into the enzyme's reaction is considered as a solution for these problems; however, native D-PhgAT is not significantly stable in such solvents. Objective: Halophiles are known to survive and withstand unsavory habitats owing to their proteome bios. In the current study, with an eye on further industrial applications, we examined the performance and thermostability of four halophilic peptides fused D-PhgAT variants in reaction mixtures of various proportions of different miscible organic solvents and various temperatures as well as desiccation. Materials and methods: Plasmid constructs from the previous study (Two alpha helixes and loops between them from Halobacterium salinarum ferredoxin enzyme fused at N-terminus domain of D-PhgAT) expressed in Escherichia coli and then D-PhgAT purified. Purified proteins were subjected to various proportions of miscible organic solvents, different temperatures, and desiccation and then performance and thermostability monitored. Results: Study confirmed increased C 50 of all halophilic fused D-PhgAT variants, where the highest C 50 observed for ALAL-D-PhgAT (30.20±2.84 %V/V). Additionally, all halophilic fused variants showed higher thermostability than the wild-type D-PhgAT in the presence of different fractions of acetone, N,N-Dimethylformamide and isopropanol in aqueous binary media, while zero activity observed at the presence of methanol. Conclusion: Our results suggest that applying this new technique could be invaluable for making enzymes durable in discordant industrial conditions.

D-phenylglycine aminotransferase (D-PhgAT) from a newly isolated soil bacterium, Pseudomonas stutzeri ST-201, was purified to electrophoretic homogeneity and characterized. The molecular weight (M(r)) of the native enzyme was estimated to be 92,000. It is composed of two subunits identical in molecular weight (M(r)) = 47,500). The isoelectric point (pI) of the native enzyme was 5.0. The enzyme catalyzed reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate was an exclusive amino group acceptor and was converted into L-glutamic acid. Neither the D- nor L-isomer of phenylalanine, tyrosine, alanine, valine, leucine, isoleucine or serine could serve as a substrate. The enzyme was most active at alkaline pH with maximum activity at pH 9-10. The temperature for maximum activity was 35-45 degrees C. The apparent K(m) values for D-phenylglycine and for 2-oxoglutarate at 35 degrees C, pH 9.5 were 1.1 and 2.4 mM, respectively. The enzyme activity was strongly inhibited by typical inhibitors of pyridoxal phosphate-dependent enzymes. Possible application of this enzyme for synthesis of enantiomerically pure D-phenylglycine was demonstrated.

Phenylglycine has proved to be a useful P2 residue in HCV NS3 protease inhibitors. A novel pi-pi-interaction between the phenylglycine and the catalytic H57 residue of the protease is postulated. We hypothesized that the introduction of a vinyl on the phenylglycine might strengthen this pi-pi-interaction. Thus, herein is presented the synthesis and inhibitory potency of a series of acyclic vinylated phenylglycine-based HCV NS3 protease inhibitors. Surprisingly, inhibitors based on both D- and L-phenylglycine were found to be effective inhibitors, with a slight preference for the d-epimers. Furthermore, prime-side alkenylic extension of the C-terminal acylsulfonamide group gave significantly improved inhibitors with potencies in the nanomolar range (approximately 35 nM), potencies which were retained on mutant variants of the protease.