Observation of two forms of conformations in the reentrant condensation of DNA.

Reentrant condensation of DNA in the presence of spermidine (SPD) is studied by gel electrophoresis (GEP). It is found that the reentrant condensation of DNA induced by SPD can produce a reentrant jamming of DNA molecules at the liquid-gel interface during GEP. However, not all the DNA are jammed at the interface indicating that there are different forms of condensed DNA. A model of condensed DNA consisting of two conformations can be used to explain the experimental observations. A phase diagram of the reentrant condensation based on the jamming states of DNA in terms of the length of DNA (L) and concentration of SPD is constructed. Furthermore, no charge inversion is observed during the reentrant transition.

Crystal structure and site-directed mutagenesis studies of N-carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft.

The N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) is used on an industrial scale for the production of D-amino acids. The crystal structure of D-NCAase was solved by multiple isomorphous replacement with anomalous scattering using xenon and gold derivatives, and refined to 1.95 A resolution, to an R-factor of 18.6 %. The crystal structure shows a four-layer alpha/beta fold with two six-stranded beta sheets packed on either side by two alpha helices. One exterior layer faces the solvent, whereas the other one is buried and involved in the tight intersubunit contacts. A long C-terminal fragment extends from a monomer to a site near a dyad axis, and associates with another monomer to form a small and hydrophobic cavity, where a xenon atom can bind. Site-directed mutagenesis of His129, His144 and His215 revealed strict geometric requirements of these conserved residues to maintain a stable conformation of a putative catalytic cleft. A region located within this cleft involving Cys172, Glu47, and Lys127 is proposed for D-NCAase catalysis and is similar to the Cys-Asp-Lys site of N-carbamoylsarcosine amidohydrolase. The homologous active-site framework of these enzymes with distinct structures suggests convergent evolution of a common catalytic mechanism.

Expression, crystallization and preliminary X-ray diffraction studies of N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter.

The Agrobacterium radiobacter CCRC 14924 N-carbamyl-D-amino-acid amidohydrolase, the enzyme used for production of D-amino acids, was overexpressed in Escherichia coli JM109. The expressed protein was crystallized by vapour diffusion using lithium sulfate as precipitant. It crystallizes in space group P21 with unit-cell parameters a = 69.8, b = 67.9 and c = 137.8 A and beta = 96.4 degrees. There are four molecules per asymmetric unit. Crystals diffract to 2.8 A resolution using a rotating-anode source at cryogenic (113 K) temperatures.