Insights into peculiar fungal LPMO family members holding a short C-terminal sequence reminiscent of phosphate binding motifs.

Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature.

Phonon-assisted electron-proton transfer in [FeFe] hydrogenases: Topological role of clusters.

[FeFe] hydrogenases are enzymes that have acquired a unique capacity to synthesize or consume molecular hydrogen (H). This function relies on a complex catalytic mechanism involving the active site and two distinct electron and proton transfer networks working in concert. By an analysis based on terahertz vibrations of [FeFe] hydrogenase structure, we are able to predict and identify the existence of rate-promoting vibrations at the catalytic site and the coupling with functional residues involved in reported electron and proton transfer networks.

Vibrational Perturbation of the [FeFe] Hydrogenase H-Cluster Revealed by CH-ADT Labeling.

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe] subcluster. A -CH- ADT labeling in the synthetic diiron precursor of [2Fe] produced isotope effects observed throughout the NRVS spectrum.

Structure-based mechanistic insights into catalysis by tRNA thiolation enzymes.

In all domains of life, ribonucleic acid (RNA) maturation includes post-transcriptional chemical modifications of nucleosides. Many sulfur-containing nucleosides have been identified in transfer RNAs (tRNAs), such as the derivatives of 2-thiouridine (sU), 4-thiouridine (sU), 2-thiocytidine (sC), 2-methylthioadenosine (msA). These modifications are essential for accurate and efficient translation of the genetic code from messenger RNA (mRNA) for protein synthesis.

Arabidopsis thaliana DGAT3 is a [2Fe-2S] protein involved in TAG biosynthesis.

Acyl-CoA:diacylglycerol acyltransferases 3 (DGAT3) are described as plant cytosolic enzymes synthesizing triacylglycerol. Their protein sequences exhibit a thioredoxin-like ferredoxin domain typical of a class of ferredoxins harboring a [2Fe-2S] cluster. The Arabidopsis thaliana DGAT3 (AtDGAT3; At1g48300) protein is detected in germinating seeds.

Pyrenoid functions revealed by proteomics in Chlamydomonas reinhardtii.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts.

Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB.

One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases.

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster.

Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA.

On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes.

The canonical CysXXXCysXXCys motif is the hallmark of the Radical-SAM superfamily. This motif is responsible for the ligation of a [4Fe-4S] cluster containing a free coordination site available for SAM binding. The five enzymes MoaA, TYW1, MiaB, RimO and LipA contain in addition a second [4Fe-4S] cluster itself bound to three other cysteines and thus also displaying a potentially free coordination site.

Electron Paramagnetic Resonance Characterization of Three Iron-Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus.

NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation.

Expression and purification of NifB proteins from aerobic and anaerobic sources.

NifB is the key protein in the biosynthesis of nitrogenase iron-molybdenum cofactor. Due to its extreme sensitivity to O2 and inherent protein instability, NifB proteins must be purified under strict anaerobic conditions by using affinity chromatography methods. We describe here the methods for NifB purification from cells of the strict aerobic nitrogen-fixing bacterium Azotobacter vinelandii, the facultative anaerobic nitrogen-fixing bacterium Klebsiella pneumoniae, and the facultative anaerobic non-nitrogen fixing bacterium Escherichia coli recombinantly expressing a nifB gene of thermophilic origin.

Purification of O2-sensitive metalloproteins.

The most dependable factor to perform successful biochemical experiments in an O2-free environment is the experience required to set up an efficient laboratory, to properly manipulate samples, to anticipate potential O2-related problems, and to maintain the complex laboratory setup operative. There is a long list of O2-related issues that may ruin your experiments. We provide here a guide to minimize these risks.

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases.

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters.

The methylthiolation reaction mediated by the Radical-SAM enzymes.

Over the past 10 years, considerable progress has been made in our understanding of the mechanistic enzymology of the Radical-SAM enzymes. It is now clear that these enzymes appear to be involved in a remarkably wide range of chemically challenging reactions. This review article highlights mechanistic and structural aspects of the methylthiotransferases (MTTases) sub-class of the Radical-SAM enzymes.

S-Adenosylmethionine-dependent radical-based modification of biological macromolecules.

Proteins and RNA molecules enjoy a variety of chemically complex post-translational and post-transcriptional modifications. The chemistry at work in these reactions, which was considered to be exclusively ionic in nature has recently been shown to depend on radical mechanisms in some cases. The overwhelming majority of these radical-based reactions are catalyzed by 'Radical-SAM' enzymes.

Identification of eukaryotic and prokaryotic methylthiotransferase for biosynthesis of 2-methylthio-N6-threonylcarbamoyladenosine in tRNA.

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine.

Post-translational modification of ribosomal proteins: structural and functional characterization of RimO from Thermotoga maritima, a radical S-adenosylmethionine methylthiotransferase.

Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P.