Reconstitution of the Final Steps in the Biosynthesis of Valanimycin Reveals the Origin of Its Characteristic Azoxy Moiety.

Valanimycin is an azoxy-containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456-hF10. While the biosynthesis of valanimycin has been partially characterized, how the azoxy group is constructed remains obscure. Herein, the membrane protein VlmO and the putative hydrazine synthetase ForJ from the formycin biosynthetic pathway are demonstrated to catalyze N-N bond formation converting O-(l-seryl)-isobutyl hydroxylamine into N-(isobutylamino)-l-serine. Subsequent installation of the azoxy group is shown to be catalyzed by the non-heme diiron enzyme VlmB in a reaction in which the N-N single bond in the VlmO/ForJ product is oxidized by four electrons to yield the azoxy group. The catalytic cycle of VlmB appears to begin with a resting µ-oxo diferric complex in VlmB, as supported by Mössbauer spectroscopy. This study also identifies N-(isobutylamino)-d-serine as an alternative substrate for VlmB leading to two azoxy regioisomers. The reactions catalyzed by the kinase VlmJ and the lyase VlmK during the final steps of valanimycin biosynthesis are established as well. The biosynthesis of valanimycin was thus fully reconstituted in vitro using the enzymes VlmO/ForJ, VlmB, VlmJ and VlmK. Importantly, the VlmB-catalyzed reaction represents the first example of enzyme-catalyzed azoxy formation and is expected to proceed by an atypical mechanism.

S-Adenosylmethionine: more than just a methyl donor.

Covering: from 2000 up to the very early part of 2023S-Adenosyl-L-methionine (SAM) is a naturally occurring trialkyl sulfonium molecule that is typically associated with biological methyltransfer reactions. However, SAM is also known to donate methylene, aminocarboxypropyl, adenosyl and amino moieties during natural product biosynthetic reactions. The reaction scope is further expanded as SAM itself can be modified prior to the group transfer such that a SAM-derived carboxymethyl or aminopropyl moiety can also be transferred. Moreover, the sulfonium cation in SAM has itself been found to be critical for several other enzymatic transformations. Thus, while many SAM-dependent enzymes are characterized by a methyltransferase fold, not all of them are necessarily methyltransferases. Furthermore, other SAM-dependent enzymes do not possess such a structural feature suggesting diversification along different evolutionary lineages. Despite the biological versatility of SAM, it nevertheless parallels the chemistry of sulfonium compounds used in organic synthesis. The question thus becomes how enzymes catalyze distinct transformations via subtle differences in their active sites. This review summarizes recent advances in the discovery of novel SAM utilizing enzymes that rely on Lewis acid/base chemistry as opposed to radical mechanisms of catalysis. The examples are categorized based on the presence of a methyltransferase fold and the role played by SAM within the context of known sulfonium chemistry.

Characterization of the coformycin biosynthetic gene cluster in Streptomyces kaniharaensis.

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-[Formula: see text]-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.

Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase.

Oxazinomycin is a C-nucleoside natural product with antibacterial and antitumor activities. In addition to the characteristic C-glycosidic linkage shared with other C-nucleosides, oxazinomycin also features a structurally unusual 1,3-oxazine moiety, the biosynthesis of which had previously been unknown. Herein, complete in vitro reconstitution of the oxazinomycin biosynthetic pathway is described. Construction of the C-glycosidic bond between ribose 5-phosphate and an oxygen-labile pyridine heterocycle is catalyzed by the C-glycosidase OzmB and involves formation of an enzyme-substrate Schiff base intermediate. The DUF4243 family protein OzmD is shown to catalyze oxygen insertion and rearrangement of the pyridine C-nucleoside intermediate to generate the 1,3-oxazine moiety along with the elimination of cyanide. Spectroscopic analysis and mutagenesis studies indicate that OzmD is a novel nonheme iron-dependent enzyme in which the catalytic iron center is likely coordinated by four histidine residues. These results provide the first example of 1,3-oxazine biosynthesis catalyzed by an unprecedented iron-dependent mono-oxygenase.

Identification of a Pyrrole Intermediate Which Undergoes C-Glycosidation and Autoxidation to Yield the Final Product in Showdomycin Biosynthesis.

Showdomycin is a C-nucleoside bearing an electrophilic maleimide base. Herein, the biosynthetic pathway of showdomycin is presented. The initial stages of the pathway involve non-ribosomal peptide synthetase (NRPS) mediated assembly of a 2-amino-1H-pyrrole-5-carboxylic acid intermediate. This intermediate is prone to air oxidation whereupon it undergoes oxidative decarboxylation to yield an imine of maleimide, which in turn yields the maleimide upon acidification. It is also shown that this pyrrole intermediate serves as the substrate for the C-glycosidase SdmA in the pathway. After coupling with ribose 5-phosphate, the resulting C-nucleoside undergoes a similar sequence of oxidation, decarboxylation and deamination to afford showdomcyin after exposure to air. These results suggest that showdomycin could be an artifact due to aerobic isolation; however, the autoxidation may also serve to convert an otherwise inert product of the biosynthetic pathway to an electrophilic C-nucleotide thereby endowing showdomycin with its observed bioactivities.

Identification of the Formycin A Biosynthetic Gene Cluster from Streptomyces kaniharaensis Illustrates the Interplay between Biological Pyrazolopyrimidine Formation and de Novo Purine Biosynthesis.

Formycin A is a potent purine nucleoside antibiotic with a C-glycosidic linkage between the ribosyl moiety and the pyrazolopyrimidine base. Herein, a cosmid is identified from the Streptomyces kaniharaensis genome library that contains the for gene cluster responsible for the biosynthesis of formycin. Subsequent gene deletion experiments and in vitro characterization of the forBCH gene products established their catalytic functions in formycin biosynthesis. Results also demonstrated that PurH from de novo purine biosynthesis plays a key role in pyrazolopyrimidine formation during biosynthesis of formycin A. The participation of PurH in both pathways represents a good example of how primary and secondary metabolism are interlinked.

Identification of the C-Glycoside Synthases during Biosynthesis of the Pyrazole-C-Nucleosides Formycin and Pyrazofurin.

C-Nucleosides are characterized by a C-C rather than a C-N linkage between the heterocyclic base and the ribofuranose ring. While the biosynthesis of pseudouridine-C-nucleosides has been studied, less is known about the pyrazole-C-nucleosides such as the formycins and pyrazofurin. Herein, genome screening of Streptomyces candidus NRRL 3601 led to the discovery of the pyrazofurin biosynthetic gene cluster pyf. In vitro characterization of gene product PyfQ demonstrated that it is able to catalyze formation of the C-glycoside carboxyhydroxypyrazole ribonucleotide (CHPR) from 4-hydroxy-1H-pyrazole-3,5-dicarboxylic acid and phosphoribosyl pyrophosphate (PRPP). Similarly, ForT, the PyfQ homologue in the formycin pathway, can catalyze the coupling of 4-amino-1H-pyrazole-3,5-dicarboxylic acid and PRPP to form carboxyaminopyrazole ribonucleotide. Finally, PyfP and PyfT are shown to catalyze amidation of CHPR to pyrazofurin 5'-phosphate thereby establishing the latter stages of both pyrazofurin and formycin biosynthesis.

Functionalization of C-H Bonds by Photoredox Catalysis.

Visible-light photoredox catalysis has been successfully used in the functionalization of inert C-H bonds including C(sp2 )-H bonds of arenes and C(sp3 )-H bonds of aliphatic compounds over the past decade. These transformations are typically promoted by the process of single-electron-transfer (SET) between substrates and photo-excited photocatalyst upon visible light irradiation (household bulbs or LEDs). Compared with other synthetic strategies, such as the transition-metal catalysis and traditional radical reactions, visible-light photoredox approach has distinct advantages in terms of operational simplicity and practicability. Versatile direct functionalization of inert C(sp2 )-H and C(sp3 )-H bonds including alkylation, trifluoromethylation, arylation and amidation, has been achieved using this practical strategy.

Visible-light-promoted chloramination of olefins with N-chlorosulfonamide as both nitrogen and chlorine sources.

A visible-light-promoted chloramination of olefins is reported. N-Chlorosulfonamides serve as both nitrogen and chlorine sources. These reactions provide a simple, efficient, regioselective, and atom-economical method for the preparation of vicinal haloamine derivatives under mild reaction conditions. A variety of olefins were tolerated, and chloramination products were obtained in good yields.

Primary cell culture of suprachiasmatic nucleus.

In mammals, the suprachiasmatic nucleus (SCN) contains a biological clock that drives circadian rhythms in vivo and in vitro. Primary dissociated neuronal culture is a useful research tool, which allows cell-by-cell morphological and physiological study of the SCN. A long-term primary dissociated SCN neuron culture is the prerequisite to understanding how neural activity and morphology interact in the SCN. The essential details of recent effective SCN culture methods are reviewed, including preparation of cells, medium and substrate, maintenance of cultures, and characterization of cultured SCN neurons. This technique is growing in importance, especially with the advent of multi-electrode array (MEA) recording.