Structure and function of the terpene biosynthesis enzyme, IspH

The protein IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate reductase, is an essential enzyme in isoprenoid biosynthesis and an important drug/herbicide target. The main research in this work investigated the structures and functions of IspHs from various organisms. Using a sequence similarity network we found that some IspHs form "Rosetta-stone" fusion proteins with either the ribosomal protein S1 (RPS1), or a UbiA (4-hydroxybenzoate octaprenyltransferase)-like protein. These fusion proteins are all from obligately anaerobic bacteria. A catalytically active IspH-RPS1 was expressed and characterized. The existance of these fusion proteins is indicative of possible secondary roles of the enzyme IspH, perhaps being involved in O2 sensing and regulation. Using crystallographic and bioinformatics results we show that IspHs can be classified into four major classes, based on the arrangement of the aromatic residues near the 4Fe-4S cluster and the presence of N- and C-terminal extensions, and these structure features are related to the environments in which IspHs are found. These aromatic groups protect the 4Fe-4S clusters from oxidation and are also involved in electron transfer. The results revealed how nature has evolved different structure features for a sensitive protein in different environments. In addition, another terpene biosyntheis enzyme, MenA, and two other metalloenzymes that are drug targets, DHAD and LOX, were also investigated. Overall, this research is of interest since it revealed the structure and functions of the terpene biosynthesis enzyme IspH from various organisms, as well as other proteins that are possible drug targets

Catalytic reduction of dinitrogen to silylamines by earth-abundant lanthanide and group 4 complexes

Dinitrogen is a challenging molecule to reduce to useful products under ambient conditions. The range of d-block metal complexes that can catalyze dinitrogen reduction to ammonia or tris(silyl)amines under ambient conditions has increased recently but lacks electropositive metal complexes, such as those of the f-block which lack filled d-orbitals that would support classical binding modes of N2. Here, metallacyclic phenolate structures with lanthanide or group 4 cations can bind dinitrogen and catalyze its conversion to bis(silyl)amines under ambient conditions. The formation of this unusual product is controlled by metallacycle sterics. The group 4 complexes featuring small cavities are most selective for bis(silyl)amine, while the lanthanide complexes and the solvated uranium(IV) congener, with larger cavities, can also make the conventional tris(silyl)amine product. These results offer new catalytic applications for plentiful titanium and the more earth-abundant members of the lanthanides that are also less toxic than many base metals used in catalysis

A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster

Electronic Structure of Two Catalytic States of the [FeFe] Hydrogenase H‑Cluster As Probed by Pulse Electron Paramagnetic Resonance Spectroscopy

The active site of the [FeFe] hydrogenase (HydA1), the H-cluster, is a 6-Fe cofactor that contains CO and CN- ligands. It undergoes several different oxidation and protonation state changes in its catalytic cycle to metabolize H2. Among them, the well-known Hox state and the recently identified Hhyd state are thought to be directly involved in H2 activation and evolution, and they are both EPR active with net spin S = 1/2. Herein, we report the pulse electronic paramagnetic spectroscopic investigation of these two catalytic states in Chlamydomonas reinhardtii HydA1 ( CrHydA1). Using an in vitro biosynthetic maturation approach, we site-specifically installed 13C into the CO or CN- ligands and 57Fe into the [2Fe]H subcluster of the H-cluster in order to measure the hyperfine couplings to these magnetic nuclei. For Hox, we measured 13C hyperfine couplings (13CO aiso of 25.5, 5.8, and 4.5 MHz) corresponding to all three CO ligands in the H-cluster. We also observed two 57Fe hyperfine couplings (57Fe aiso of ∼17 and 5.7 MHz) arising from the two Fe atoms in the [2Fe]H subcluster. For Hhyd, we only observed two distinct 13CO hyperfine interactions (13CO aiso of 0.16 and 0.08 MHz) and only one for 13CN- (13CN aiso of 0.16 MHz); the couplings to the 13CO/13CN- on the distal Fe of [2Fe]H may be too small to detect. We also observed a very small (<0.3 MHz) 57Fe HFI from the labeled [2Fe]H subcluster and four 57Fe HFI from the labeled [4Fe-4S]H subcluster (57Fe aiso of 7.2, 16.6, 28.2, and 35.3 MHz). These hyperfine coupling constants are consistent with the previously proposed electronic structure of the H-cluster at both Hox and Hhyd states and provide a basis for more detailed analysis