Conserved Enzymatic Cascade for Bacterial Azoxy Biosynthesis

Shi, Jingkun; Zang, Xin; Zhao, Zhijie; Shen, Zhuanglin; Li, Wei; Zhao, Guiyun; Zhou, Jiahai; Du, Yi-Ling

doi:10.1021/jacs.3c12018

Cited by 6 publications

(8 citation statements)

References 46 publications

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“…We did not observe oxidation of these hydrazine and azo compounds in the mutant, suggesting that the AzdB is required to produce the azoxy group and carries out the oxidation reaction of the hydrazine/azo group. With recent in vitro characterization , of the AzdB homologue VlmB, these findings further confirmed that AzdB is a nonheme diiron enzyme responsible for conversion of the hydrazine into the azoxy compound. The in vitro work on the biochemistry of VlmB , clearly shows that the enzyme is capable of converting hydrazine into azoxy molecule.…”

Section: Resultssupporting

confidence: 54%

“…The AzdO homologue VlmO was recently characterized to catalyze the intramolecular arrangement into a hydrazine product . No intermediates could be detected in the azdA STOP and azdO STOP mutants, likely due to the formation of an unstable pathway intermediate …”

Section: Resultsmentioning

confidence: 99%

“…The in vitro work on the biochemistry of VlmB , clearly shows that the enzyme is capable of converting hydrazine into azoxy molecule. The unexpected presence of the azo compounds 10 – 12 in the azdB STOP mutant indicates that either AzdB is not required for the formation of the azo molecule in the mutant strain (which would not be in line with the thorough biochemical analysis of the close homologue vlmB), it is catalyzed by a yet unidentified enzyme encoded outside of the azd -BGC, or, most likely, it is readily formed under the aerobic culture conditions …”

Section: Resultsmentioning

confidence: 99%

“…Azoxy compounds have gained a considerable interest in the past four years. ,,,,, However, there is still limited knowledge about key biochemical steps and functions of the individual genes and BGCs. In this study, we confirmed the BGC of azodyrecins through heterologous expression and extensive mutational analysis of the azd BGC using CRISPR-Cas9 base editing.…”

Section: Discussionmentioning

confidence: 99%

“…At the very end, the valanimycin hydrate is phosphorylated followed by dehydration by VlmJ and VlmK. The three genes, vlmB , vlmG , and vlmO , remained enigmatic until recent work performed by Zheng et al and Shi et al, who investigated the azoxy bond formation through in vitro enzyme assays and other approaches. The studies confirmed that vlmO encodes transmembrane protein that, together with VlmL, seryl tRNA synthetase, , and VlmA, catalyzes the intramolecular arrangement into a hydrazine product.…”

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Maleckis,

Wibowo,

Gren

et al. 2024

ACS Chem. Biol.

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Azoxy compounds are a distinctive group of bioactive secondary metabolites characterized by a unique RN� N + (O − )R moiety. The azoxy moiety is present in various classes of metabolites that exhibit various biological activities. The enzymatic mechanisms underlying azoxy bond formation remain enigmatic. Azodyrecins are cytotoxic azoxy metabolites produced by Streptomyces mirabilis P8-A2. Here, we cloned and confirmed the putative azd biosynthetic gene cluster through CATCH cloning followed by expression and production of azodyrecins in two heterologous hosts, S. albidoflavus J1074 and S. coelicolor M1146, respectively. We explored the function of 14 enzymes in azodyrecin biosynthesis through gene knockout using CRISPR-Cas9 base editing in the native producer, S. mirabilis P8-A2. The key intermediates were analyzed in the mutants through MS/MS fragmentation studies, revealing azoxy bond formation via the conversion of hydrazine to an azo compound followed by further oxygenation. Enzymes involved in modifications of the precursor could be postulated based on their predicted function and the intermediates identified in the knockout strains. Moreover, the distribution of the azoxy biosynthetic gene clusters across Streptomyces spp. genomes is explored, highlighting the presence of these clusters in over 20% of the Streptomyces spp. genomes and revealing that azoxymycin and valanimycin are scarce, while azodyrecin and KA57A-like clusters are widely distributed across the phylogenetic tree.

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Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Maleckis,

Wibowo,

Gren

et al. 2024

ACS Chem. Biol.

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Diverse N‐Oxidation of Primary Aromatic Amines Controlled by Engineered P450 Peroxizyme Variants Facilitated by Dual‐Functional Small Molecule

Chen,

Yao,

Jiang

et al. 2024

Advanced Science

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Amine oxidation is an important organic reaction for the production of high‐value N‐containing compounds. However, it is still challenging to control the reactivity of active N‐centered radicals to selectively access N‐oxidation products. Herein, this study reports the engineering of cytochrome P450BM3 into multifunctional N‐oxidizing enzymes with the assistance of dual‐functional small molecules (DFSM) to selectively produce N‐oxygenation (i.e., p‐nitrosobenzene, p‐nitrobenzene, and azoxybenzene) and one‐electron oxidation products (i.e., oligomeric quinones and azobenzene) from aromatic amines. The best mutant, F87A/T268V/V78T/A82T, exclusively gives p‐nitrosobenzene (up to 98% selectivity), whereas the selectivity for p‐nitrobenzene is >99% using the mutant F87A/T268V/A82T/I263L. Crystal structure analysis reveals that key mutations and DFSM exert synergistic effects on catalytic promiscuity by controlling the substrate orientation in active center. This study highlights the potential of DFSM‐facilitated P450 peroxygenase and peroxidase for the synthesis of N‐containing compounds via the controllable oxidation of aromatic amines, substantially expanding the chemical space of P450 enzymes.

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Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

Matsuda,

Nakahara,

Choirunnisa

et al. 2024

ChemBioChem

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Cupin/methionyl‐tRNA synthetase (MetRS)‐like didomain enzymes catalyze nitrogen‐nitrogen (N–N) bond formation between Nw‐hydroxylamines and amino acids to generate hydrazines, key biosynthetic intermediates of various natural products containing N–N bonds. While the combination of these two building blocks leads to the creation of diverse hydrazine products, the full extent of their structural diversity remains largely unknown. To explore this, we herein conducted phylogeny‐guided genome‐mining of related hydrazine biosynthetic pathways consisting of two enzymes: flavin‐dependent Nw‐hydroxylating monooxygenases (NMOs) that produce Nw‐hydroxylamine precursors and cupin/MetRS‐like enzymes that couple the Nw‐hydroxylamines with amino acids via N–N bonds. A phylogenetic analysis identified the largely unexplored sequence spaces of these enzyme families. The biochemical characterization of NMOs demonstrated their capabilities to produce various Nw‐hydroxylamines, including those previously not known as precursors of N–N bonds. Furthermore, the characterization of cupin/MetRS‐like enzymes identified five new hydrazine products with novel combinations of building blocks, including one containing non‐amino acid building blocks: 1,3‐diaminopropane and putrescine. This study substantially expanded the variety of N–N bond forming pathways mediated by cupin/MetRS‐like enzymes.

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Conserved Enzymatic Cascade for Bacterial Azoxy Biosynthesis

Cited by 6 publications

References 46 publications

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Biosynthesis of the Azoxy Compound Azodyrecin from Streptomyces mirabilis P8-A2

Diverse N‐Oxidation of Primary Aromatic Amines Controlled by Engineered P450 Peroxizyme Variants Facilitated by Dual‐Functional Small Molecule

Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

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