Cytochrome P450 enzymes are highly diversified biocatalysts associated with steroid biosynthesis, xenobiotic metabolism, biosynthesis of natural products, and industrial oxidation reactions. A typical P450 catalytic cycle requires sequential transfer of two electrons from NAD(P)H to the heme-iron reactive center for O 2 activation. For the most abundant bacterial Class I P450 systems, this important process is usually mediated by two redox partner proteins including an FAD-containing ferredoxin reductase (FdR) and a small iron−sulfur protein, ferredoxin (Fdx). However, it is often unclear which pair of Fdx and FdR among multiple redox partners is the optimal one for a specific Class I P450 enzyme. To address this important but underexplored question, herein, a reaction matrix network with 16 Fdxs, 8 FdRs, and 6 P450s (against 7 substrates) was constituted. By analyzing the reactivity profiles of 896 P450 reactions, together with phylogenetic analysis, redox potential measurements, structural simulations, and Fdx-P450 molecular docking, we provide important mechanistic insights into the recognition and interactions between bacterial Class I P450 enzymes and redox partners.
The C-10–C-4a bond cleavage
of anthraquinone is believed
to be a crucial step in fungal seco-anthraquinone biosynthesis and
has long been proposed as a classic Baeyer–Villiger oxidation.
Nonetheless, genetic, enzymatic, and chemical information on ring
opening remains elusive. Here, a revised questin ring-opening mechanism
was elucidated by in vivo gene disruption, in vitro enzymatic analysis, and 18O chasing
experiments. It has been confirmed that the reductase GedF is responsible
for the reduction of the keto group at C-10 in questin to a hydroxyl
group with the aid of NADPH. The C-10–C-4a bond of the resultant
questin hydroquinone is subsequently cleaved by the atypical cofactor-free
dioxygenase GedK, giving rise to desmethylsulochrin. This proposed
bienzyme-catalytic and dioxygenation-mediated anthraquinone ring-opening
reaction shows universality.
Plant-parasitic nematodes (PPNs) cause serious harm to agricultural production. Bacillus firmus shows excellent control of PPNs and has been produced as a commercial nematicide. However, its nematicidal factors and mechanisms are still unknown. In this study, we showed that B. firmus strain DS-1 has high toxicity against Meloidogyne incognita and soybean cyst nematode. We sequenced the whole genome of DS-1 and identified multiple potential virulence factors. We then focused on a peptidase S8 superfamily protein called Sep1 and demonstrated that it had toxicity against the nematodes Caenorhabditis elegans and M. incognita. The Sep1 protein exhibited serine protease activity and degraded the intestinal tissues of nematodes. Thus, the Sep1 protease of B. firmus is a novel biocontrol factor with activity against a root-knot nematode. We then used C. elegans as a model to elucidate the nematicidal mechanism of Sep1, and the results showed that Sep1 could degrade multiple intestinal and cuticle-associated proteins and destroyed host physical barriers. The knowledge gained in our study will lead to a better understanding of the mechanisms of B. firmus against PPNs and will aid in the development of novel bio-agents with increased efficacy for controlling PPNs.
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