Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. The biosynthesis of the lower ligand of vitamin B(12), 5,6-dimethylbenzimidazole (DMB), is poorly understood. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and D-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.

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The antitumor fungal metabolite terrequinone A, identified in extracts of Aspergillus sp., is biosynthesized by the five-gene cluster tdiA-tdiE. In this work, we have overproduced all five proteins (TdiA-TdiE) in the bacterial host Escherichia coli, fully reconstituting the biosynthesis of terrequinone A. This pathway involves aminotransferase activity, head-to-tail dimerization and bisprenylation of the scaffold to yield the benzoquinone natural product. We have established that TdiD is a pyridoxal-5'-phosphate-dependent L-tryptophan aminotransferase that generates indolepyruvate for an unusual nonoxidative coupling by the tridomain nonribosomal peptide synthetase TdiA. TdiC, an NADH-dependent quinone reductase, generates the nucleophilic hydroquinone for two distinct rounds of prenylation by the single prenyltransferase TdiB. TdiE is required to shunt the benzoquinone away from an off-pathway monoprenylated species by an as yet unknown mechanism. Overall, we have biochemically characterized the complete biosynthetic pathway to terrequinone A, highlighting the nonoxidative dimerization pathway and the unique asymmetric prenylation involved in its maturation.

In the biosynthesis of the antitumor indolocarbazoles rebeccamycin and staurosporine by streptomycetes, assembly of the aglycones involves a complex set of oxidative condensations. Overall formation of aglycones K252c and arcyriaflavin A from their biosynthetic precursor chromopyrrolic acid involves four- and eight-electron oxidations, respectively. This process is catalyzed by the remarkable enzyme StaP, with StaC and RebC acting to direct the level of oxidation in the newly formed five-membered ring. An aryl-aryl coupling reaction is integral to this transformation as well as oxidative decarboxylation of the dicarboxypyrrole moiety of chromopyrrolic acid. Herein we describe the heterologous expression of staP, staC, and rebC in Escherichia coli and the activity of the corresponding enzymes in constructing the two distinct six-ring scaffolds. StaP is a cytochrome P450 enzyme, requiring dioxygen, ferredoxin, flavodoxin NADP(+)-reductase, and NAD(P)H for activity. StaP on its own converts chromopyrrolic acid into three aglycone products, K252c, arcyriaflavin A, and 7-hydroxy-K252c; in the presence of StaC, K252c is the predominant product, while the presence of RebC directs formation of arcyriaflavin A. (18)O-Labeling studies indicate that the oxygen(s) of the pyrrolinone and maleimide functionalities of the aglycones formed are all derived from dioxygen. This work allowed for the in vitro reconstitution of the full biosynthetic pathway from l-tryptophan to the staurosporine and rebeccamycin aglycones, K252c and 1,11-dichloroarcyriaflavin A.

The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron oxidation of chlorinated chromopyrrolic acid. Although one rebeccamycin biosynthetic enzyme is capable of generating low levels of the eight-electron oxidation product on its own, a second protein, RebC, is required to accelerate product formation and eliminate side reactions. However, the mode of action of RebC was largely unknown. Using crystallography, we have determined a likely function for RebC as a flavin hydroxylase, captured two snapshots of its dynamic catalytic cycle, and trapped a reactive molecule, a putative substrate, in its binding pocket. These studies strongly suggest that the role of RebC is to sequester a reactive intermediate produced by its partner protein and to react with it enzymatically, preventing its conversion to a suite of degradation products that includes, at low levels, the desired product.flavin enzymes ͉ x-ray crystallography ͉ indolocarbazoles ͉ antitumor R ebeccamycin (1, Fig. 1a) is a natural product isolated from Lechevalieria aerocolonigenes (1) and is a prototype for a class of compounds that bind to DNA-topoisomerase I complexes (2), preventing the replication of DNA and thereby acting as antitumor compounds (3). Rebeccamycin is synthesized by the action of eight enzymes, with the overall conversion of Ltryptophan, chloride, molecular oxygen, glucose, and a methyl group to the glycosylated indolocarbazole rebeccamycin (1) (4-9). One step in this pathway is the conversion of chlorinated chromopyrrolic acid (2) to the rebeccamycin aglycone (3); this reaction involves an eight-electron oxidation, and it is carried out by the enzyme pair RebP and RebC (10). The mechanism of this reaction is not established (8).RebP is annotated as a cytochrome P450, and it is functionally equivalent to its homologue StaP from the staurosporine biosynthetic pathway (10), which has been used in place of RebP in initial biochemical investigations. Additionally, all biochemical work on StaP and RebC has used nonchlorinated substrates because they are well tolerated by both enzymes (8). One of the most remarkable features of StaP is that when it is incubated alone with its nonchlorinated substrate, chromopyrrolic acid (4), as well as with an electron source (provided by ferredoxin, flavodoxin NADP ϩ -reductase, and NAD(P)H), StaP is capable of generating a number of products, including arcyriaflavin A (5), 7-hydroxy-K252c (6), and K252c (7) at a 1:7:1 ratio (8) (Fig. 1b). Inclusion of RebC, an FAD-containing protein, in the reaction mixture results in near-exclusive production of arcyriaflavin A (5) and acceleration of turnover (8). One possibility to account for this phenomenon is that RebC can direct the outcome of the reaction by mediating the catalytic activity of StaP through a protein-protein interaction rather than acting catalytically itself. Although no stable interaction was found between StaP and RebC through pull-down assays (8), a transient complex cannot be ruled out. Another possibility...

During the biosynthesis of the fused six-ring indolocarbazole scaffolds of rebeccamycin and staurosporine, two molecules of L-tryptophan are processed to a pyrrole-containing five-ring intermediate known as chromopyrrolic acid. We report here the heterologous expression of RebO and RebD from the rebeccamycin biosynthetic pathway in Escherichia coli, and tandem action of these two enzymes to construct the dicarboxypyrrole ring of chromopyrrolic acid. Chromopyrrolic acid is oxidized by six electrons compared to the starting pair of L-tryptophan molecules. RebO is an L-tryptophan oxidase flavoprotein and RebD a heme protein dimer with both catalase and chromopyrrolic acid synthase activity. Both enzymes require dioxygen as a cosubstrate. RebD on its own is incompetent with L-tryptophan but will convert the imine of indole-3-pyruvate to chromopyrrolic acid. It displays a substrate preference for two molecules of indole-3-pyruvic acid imine, necessitating a net two-electron oxidation to give chromopyrrolic acid.Rebeccamycin 1 and staurosporine 2 ( Figure 1) are indolocarbazole antibiotics originally isolated from the actinomycetes LecheValieria aerocolonigenes and Streptomyces longisporoflaVus, respectively. Rebeccamycin is an inhibitor of DNA topoisomerase I, with a minimum inhibitory concentration of 1.75 µM (1). Staurosporine is one of the strongest inhibitors of protein kinases, displaying an IC 50 of 2.7 nM for protein kinase C (2) and IC 50 's in the range of 1-20 nM for most protein kinases. Because of the importance of both the protein kinases and DNA topoisomerases in cell growth and proliferation, these two compounds have been extensively studied as antitumor drug candidates. Analogues of both rebeccamycin and staurosporine have entered clinical trials for the treatment of neoplastic tumors (3, 4), renal cell cancer (5), and leukemia (6).The fused six-ring indolocarbazole scaffold in 1 and 2 is representative of a variety of natural products and derived from the dimerization of two molecules of L-tryptophan (LTrp) 1 via a complex set of oxidative transformations (7-10) (Scheme 1). Since this natural product scaffold is featured in molecules with an interesting range of biological activities, there has been substantial interest in understanding the enzymology of the oxidative dimerization and ring fusion from the starting pair of L-Trp substrates. Subsequent N-glycosylation of the aglycone through the anomeric carbon of a glucose moiety (in the case of rebeccamycin) and through both C 1 and C 5 of L-ristosamine, the deoxyhexose sugar of staurosporine, followed by the action of methyltransferases (11), leads to the active natural products (Scheme 1). In the case of staurosporine, the enzymatic generation of the N-C linkage between one indole nitrogen and C5 of the aminodeoxyhexose is of notable interest as a novel transformation (11,12).The biosynthetic gene clusters for rebeccamycin and staurosporine have been sequenced and functionally expressed in the heterologous host Escherichia coli, validatin...

As the SARS-CoV-2 pandemic unfolds across the globe, consistent themes are emerging with regard to aspects of SARS-CoV-2 infection and its associated disease entities in children. Overall, children appear to be less frequently infected by, and affected by, SARS-CoV-2 virus and the clinical disease COVID-19. Large epidemiological studies have revealed children represent less than 2% of the total confirmed COVID-19 cases, of whom the majority experience minimal or mild disease that do not require hospitalisation. Children do not appear to be major drivers of SARS-CoV-2 transmission, with minimal secondary virus transmission demonstrated within families, schools and community settings. There are several postulated theories regarding the relatively low SARS-CoV-2 morbidity and mortality seen in children, which largely relate to differences in immune responses compared to adults, as well as differences in angiotensin converting enzyme 2 distribution that potentially limits viral entry and subsequent inflammation, hypoxia and tissue injury. The recent emergence of a multisystem inflammatory syndrome bearing temporal and serological plausibility for an immunemediated SARS-CoV-2-related disease entity is currently under investigation. This article summarises the current available data regarding SARS-CoV-2 and the paediatric population, including the spectrum of disease in children, the role of children in virus transmission, and host-virus factors that underpin the unique aspects of SARS-CoV-2 pathogenicity in children.

Children globally have been profoundly impacted by the coronavirus disease 2019 (COVID‐19) pandemic. This review explores the direct and indirect public health impacts of COVID‐19 on children. We discuss in detail the transmission dynamics, vaccination strategies and, importantly, the ‘shadow pandemic’, encompassing underappreciated indirect impacts of the pandemic on children. The indirect effects of COVID‐19 will have a long‐term impact beyond the immediate pandemic period. These include the mental health and wellbeing risks, disruption to family income and attendant stressors including increased family violence, delayed medical attention and the critical issue of prolonged loss of face‐to‐face learning in a normal school environment. Amplification of existing inequities and creation of new disadvantage are likely additional sequelae, with children from vulnerable families disproportionately affected. We emphasise the responsibility of paediatricians to advocate on behalf of this vulnerable group to ensure the longer‐term effects of COVID‐19 public health responses on the health and wellbeing of children are fully considered.