The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron-and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C222 1 ), consistent with solution studies. CarC monomers contain a double-stranded ␤-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with L-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis.

The influenza virus RNA-dependent RNA polymerase protein complex contains an associated RNA endonuclease activity, which cleaves host mRNA precursors in the cell nucleus at defined positions 9-15 nucleotides downstream of the cap structure. This reaction provides capped oligoribonucleotides, which function as primers for the initiation of viral mRNA synthesis. The endonuclease reaction is dependent on the presence of divalent metal ions. We have used a number of divalent and trivalent metal ions alone and in combination to probe the mechanism of RNA cleavage by the influenza virus endonuclease. Virus-specific cleavage was observed with various metal ions, and maximum cleavage activity was obtained with 100 microM Mn2+ or 100 microM Co2+. This activity was about 2-fold higher than that observed with Mg2+ at the optimal concentration of 1 mM. Activity dependence on metal ion concentration was cooperative with Hill coefficients close to or larger than 2. Synergistic activation of cleavage activity was observed with combinations of different metal ions at varying concentrations. These results support a two-metal ion mechanism of RNA cleavage for the influenza virus cap-dependent endonuclease. The findings are also consistent with a structural model of the polymerase, in which the specific endonuclease active site is spatially separated from the nucleotidyl transferase active site of the polymerase module.

Traditional successful antiviral vaccines have relied mostly on live-attenuated viruses. Live-attenuated HIV vaccine candidates are not ideal as they pose risks of reversion, recombination or mutations. Other current HIV vaccine candidates have difficulties generating broadly effective neutralising antibodies and cytotoxic T cell immune responses to primary HIV isolates. Virus-like-particles (VLPs) have been demonstrated to be safe to administer to animals and human patients as well as being potent and efficient stimulators of cellular and humoral immune responses. Therefore, VLPs are being considered as possible HIV vaccines. Chimeric HIV-1 VLPs constructed with either HIV or SIV capsid protein plus HIV immune epitopes and immuno-stimulatory molecules have further improved on early VLP designs, leading to enhanced immune stimulation. The administration of VLP vaccines via mucosal surfaces has also emerged as a promising strategy with which to elicit mucosal and systemic humoral and cellular immune responses. Additionally, new information on antigen processing and the presentation of particulate antigens by dendritic cells (DCs) has created new strategies for improved VLP vaccine candidates. This paper reviews the field of HIV-1 VLP vaccine development, focusing on recent studies that will likely uncover promising prospects for new HIV vaccines.

During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding.

Electrochemistry of self-assembled DNA monolayers represents an attractive strategy for understanding the intrinsic properties of DNA and for developing DNA-based sensors. Thus, there is much interest in the discovery and characterization of new redox-active probes for application in DNA-based technologies. Herein, we report a detailed study of the electrochemical properties of a perylene-3,4,9,10-tetracarboxylic diimide base surrogate, when incorporated at various positions within a DNA monolayer. We demonstrate that the redox chemistry of this perylenediimide probe is mediated by the DNA base pair stack, dependent on its location within the DNA monolayer, and activated thermally. The electrochemical features and general synthetic flexibility of the perylenediimide base surrogate appear favorable for assays that leverage DNA-mediated charge transport.