4-Membered metallodithiophosphinate rings - flat or puckered?

Abstract

N-carbamoyl-D-amino-acid amidohydrolase is an industrial biocatalyst to hydrolyze N-carbamoyl D-amino acids for producing valuable D-amino acids. The crystal structure of N-carbamoyl-Damino-acid amidohydrolase in the unliganded and lingaded forms demonstrate a tetramer with-ß-ß-fold and a C172-E47-K127 catalytic triad. Crucial binding residues N173, R175, and R176 are also identified. Four mutants were further generated to engineer enzymes with additional intermolecular disulfide bridges: P178C at helix 6, A222C at helix 8, P295C/F304C and A302C from the Cterminal segment near a 2-fold axis. A302C and P295C/F304C showed an increase of 8.8°C and 3.7°C respectively in apparent melting temperature than that of the wild-type enzyme, while there was hardly any change for P178C and A222C. Crystal structures of A222C and A302C were determined and showed limited conformational change. An intermolecular disulfide bridge was observed in A302C but not in A222C. Enzymatic kinetic analysis of A302C revealed a 1.5-fold enhancement in k cat / K m at 55°C and 4.2fold increase at 65°C. Our results suggest that introducing an intermolecular disulfide bridge at the C-terminal segment of Ncarbamoyl-D-amino-acid amidohydrolase near a dyad axis is a useful approach for enhanced thermostability.

(S)-4-(4-Mercaptophenyl)-2,2,4-trimethylchroman

“…For related literature, see: Brienne & Jacques (1975); Esterhuysen et al (2005); Finocchearo & Failla (1996); Hardy et al (1977); Jacobs et al (2006); ; .…”

“…In conjunction with our investigation on the formation of clathrates (Esterhuysen et al, 2005; we have synthesized an enantiomerically pure form of 4-p-mercaptophenyl-2,2,4-trimethylchroman, (I), the thiol derivative of Dianin's compound.…”

S-[4-(2,2,4-Trimethylchroman-4-yl)phenyl]N,N-dimethylthiocarbamate

“…Dianin's compound, 4-p-hydroxyphenyl-2,2,4-trimethylchroman, is part of our research focus in the field of crystal engineering (Esterhuysen et al, 2005;. During the conversion of Dianin's compound to its thiol deriviative, the racemic mixture of O-[4-(2,2,4-trimethylchroman-4-yl)phenyl] N,N-dimethylthiocarbamate, (II), was revealed to have an interesting crystal structure that was reported by us (de Vries et al, 2005).…”