Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host.

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

Hybrid Amyloid-Based Redox Hydrogel for Bioelectrocatalytic H2 Oxidation.

An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing the hydrogenase under mild conditions at the surface of carbon electrodes were obtained by a simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H2 were fabricated that afforded catalytic current densities of up to 270 µA cm-2 , with an overpotential of 0.33 V, under quiescent conditions at 45 °C.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C3 methylthiolation of the D89 residue in the ribosomal S12 protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS- ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

Automatic detection of cirrhosis in hospitalized patients: a pragmatic experience.

BACKGROUND/AIM: We evaluated the relevance of a systematic automatic detection of cirrhosis using biochemical markers in hospitalized patients. METHODS: We automatically calculated three free biochemical tests (APRI, Fib-4, and Forns) in patients consecutively hospitalized in our university hospital between July and September, 2010. Patients >18 years not known to suffer from chronic liver disease, were contacted to undergo liver stiffness measurement (LSM) as a reference diagnostic tool. To limit false positives, we required at least one APRI≥2 (indicating cirrhosis) and Fib-4>3.25 and/or Forns>6.9, without obvious overestimation. RESULTS: A total of 10,035 APRI, 9903 Fib-4, and 1250 Forns were available in 4074 patients. The fibrosis tests were independently influenced by the location of the patient, especially Cardiology (Lower Forns) and Hematology/Oncology Departments (higher APRI, Fib-4, and Forns). Overall, 101 patients (2.48%) were suspected to have cirrhosis. LSM identified two cases of cirrhosis (LSM>13 kPa). In intent-to-diagnose analyses, the highest positive predictive values of the APRI, Fib-4, and Forns for the diagnosis of cirrhosis were 1.98, 1.98, and 11.76%, respectively. The positive predictive value never exceeded 50% in per-protocol analyses when considering patients with numerous positive results of the fibrosis tests. CONCLUSION: In hospitalized patients, automatic detection of cirrhosis on the basis of APRI, Fib-4, and Forns was inefficient because of too many false-positive results.