In anaerobic organisms such as E. coli the tyrosine lyase ThiH is essential for the biosynthesis of the thiazole moiety of the vitamin thiamine. ThiH is a member of the “radical AdoMet” family. The products formed by cleavage of tyrosine in vitro have been identified and suggest a radical‐mediated cleavage resulting in p‐cresol and dehydroglycine which is hydrolyzed to glyoxylate.

An emerging treatment for Parkinson's disease ( PD ) is cell replacement therapy. Authentic midbrain dopaminergic ( mDA ) neuronal precursors can be differentiated from human embryonic stem cells ( hESC s) and human induced pluripotent stem cells ( iPSC s). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD . However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR /Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA +/− and SNCA −/− cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils ( PFF s) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein ( pS 129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS 129‐αSyn, while SNCA +/− and SNCA −/− neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR /Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology.

Lipoyl synthase (LipA) is required for the final step in the biosynthesis of lipoyl groups, the insertion of sulfur atoms at C6 and C8 of octanoyl groups (Scheme 1).[1] The octanoyl groups are found attached through an amide linkage to a lysine residue in a sequence motif that is conserved within a small family of protein domains [2] that include the H-protein of the glycine cleavage system [3] and the E2 subunit of oxoacid dehydrogenases. [4] After attachment of the octanoyl groups, [5][6][7] the substrates undergo sulfur insertion by LipA, [1] a member of the "radical S-adenosyl l-methionine" family.[8] Biochemical studies have shown that it contains two essential [4Fe-4S] clusters [9] and that one of these clusters is used to generate 5'-deoxyadenosyl radicals (AdoC) through the reductive cleavage of S-adenosyl l-methionine (AdoMet). The lipoyl forming reaction requires two hydrogen atoms to be removed from the octanoyl group, one from C6 and the other from C8, so that the overall reaction utilizes two equivalents of AdoMet to form each lipoyl group. [10] LipA has been shown to donate both of the inserted sulfur atoms [11] that were proposed to originate from a [4Fe-4S] cluster.[12] Recently, we reported that a short octanoyl peptide, which corresponds in sequence to the lipoylation site of the E2 subunit, could function as a substrate for LipA.[13] Peptide substrates have now been used to investigate the order of sulfur-insertion steps and hence clarify the structure of a key intermediate on the reaction pathway.The formation of lipoyl groups was monitored in a reaction by using Sulfolobus solfataricus P2 LipA with an octanoyl substrate tripeptide (0.5 mole equiv; 3, ), AdoMet, and dithionite as a reductant. LipA is not catalytic during assays in vitro, producing substoichiometric quantities of lipoyl products with either octanoyl-protein [10] or octanoylpeptide substrates.[13] To investigate the formation of any intermediate species, the reaction was stopped by acidification before reaching completion (after 20 min), the precipitated protein was pelleted by centrifugation, and the supernatant analyzed by LCMS. Four peptide species were eluted over the time range from 22 to 25 min (Table 1). The peptide at 24.4 min corresponds to the unreacted octanoyl substrate 3, whereas new species at 22.4 and 23.4 min correspond to the expected protonated product masses of lipoyl 5 and dihydrolipoyl products 4, respectively. The final species at 23.2 min coelutes with the dihydrolipoyl product and corresponds to a monothiolated species (either 6 or 7). Analysis of later time points (up to 2 h) showed that the amount of this monothiolated species decreased with time and that there was a Scheme 1. Insertion of sulfur atoms into octanoyl substrates to form lipoyl groups.

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (ϳ10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

Edited by Barry HalliwellKeywords: Iron-sulfur cluster S-adenosylmethionine Enzyme kinetics Product inhibition a b s t r a c t Members of the radical S-adenosylmethionine (AdoMet) superfamily reductively cleave AdoMet to generate the highly reactive 5 0 -deoxyadenosyl radical (DOA Å ) which initiates biological transformations by abstraction of a hydrogen atom. We demonstrate that three members of the family: biotin synthase (BioB), lipoyl synthase (LipA) and tyrosine lyase (ThiH) are inhibited in vitro by a combination of the products 5 0 -deoxyadenosine (DOA) and methionine. These results suggest the observed inhibition is a common feature of the radical AdoMet proteins that form DOA and methionine as products. Addition of 5 0 -methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) to BioB, LipA or ThiH activity assays removed the product inhibition by catalysing the hydrolysis of DOA and gave an increase in activity.