Biology subjects

Du, Y.-L.

Publications and source records attributed to Du, Y.-L..

5 recordsLinked to original sources

bioRxiv2025

Structural and mechanistic insights into bacterial hydrazine biosynthesis

Nitrogen-nitrogen (N-N) bond-containing motifs are prevalent in both clinical drugs and natural products. Bacterial hydrazine synthetases catalyze N-N bond formation by coupling an amino acid and a hydroxylamine via a distinctive O-aminoacyl-hydroxylamine intermediate. Despite its wide occurrence, the structural and mechanistic basis of this process has remained elusive. Here, we report the first crystal structures of the O-aminoacyl-hydroxylamine synthetase component of a bacterial hydrazine synthetase, captured in binary and ternary complexes with substrates and catalytic intermediates. These structures reveal the molecular determinants of substrate recognition and, together with biochemical and computational analyses, establish a detailed mechanistic framework for the hydrazine synthetase family. Leveraging these insights, we expanded hydrazine biosynthesis through the targeted discovery of novel hydrazine synthetases and implemented a chemoenzymatic synthesis strategy. This work provides fundamental insights into hydrazine synthetases and lays the groundwork for their rational engineering as versatile biocatalysts.

microbiology↗

bioRxiv2025

Genome Mining and Characterization of a Heme-Dependent Enzyme Catalyzing Intermolecular Nitrogen-Nitrogen Bond Formation in Hydrazinosuccinic Acid Biosynthesis

Nitrogen-nitrogen (N-N) bond-forming enzymes are rare but play vital roles in both primary and secondary metabolism. Guided by a nitric oxide synthase (NOS)-based genome mining strategy, we report the discovery and characterization of a new heme-dependent enzyme system that catalyzes intermolecular N-N bond formation. Using both in vivo and in vitro reconstitution approaches, we demonstrated that a protein complex, comprising a heme enzyme and a 2[4Fe-4S] ferredoxin partner, mediates the coupling of the -amine group of L-aspartate with inorganic nitrogen oxide species, such as nitrite or nitric oxide, to generate hydrazinosuccinic acid, a key biosynthetic precursor in several natural product pathways. Structural modeling and site-directed mutagenesis suggest a plausible catalytic mechanism involving the formation of a reactive nitrogen intermediate, potentially a heme-bound nitrene species. These findings reveal a new family of N-N bond-forming biocatalysts that leverage inorganic nitrogen sources, offering valuable tools for genome mining and the synthetic biology.

biochemistry↗

bioRxiv2023

Structural and mechanistic views of enzymatic, heme-dependent nitrogen-nitrogen bond formation

Molecules with nitrogen-nitrogen (N-N) bonds constitute a large group of clinically important drugs, and various synthetic approaches have been developed to construct functional groups like hydrazines, diazos, pyrazoles, and N-nitrosos. While hundreds of N-N-containing specialized natural metabolites have also been discovered, little is known about the underlying enzymatic mechanisms that have evolved for N-N bond formation. In order to directly form a single N(sp3)-N(sp3) bond, enzymes must reverse the typical nucleophilicity of one nitrogen. Here, we report structural and mechanistic interrogations of the piperazate synthase PipS, a heme-dependent enzyme that catalyzes an N-N bond forming cyclization of N5-OH-L-ornithine to give the non-proteinogenic amino acid L-piperazic acid. We show that PipS can process a variety of N-substituted hydroxylamines, to give either an imine or an N-N bond, in a substrate-specific manner. Using a combination of structural and biochemical experiments, computational studies, and spectroscopic characterization, we propose that heme-dependent dehydration and N-N bond formation in PipS proceed through divergent pathways, which may stem from a shared nitrenoid intermediate that effectively reverses the nucleophilicity of the hydroxylamine nitrogen. Our work expands the current knowledge of enzymatic N-N bond formation, and delineates the catalytic versatility of a heme cofactor, paving the way for future development of genetically encoded biocatalysts for N-N bond formation.

biochemistry↗

bioRxiv2020

Expansion of gamma-butyrolactone signaling molecule biosynthesis to phosphotriester natural products

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their anti-malarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the saliniphostins and newly identified A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed amongst many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

biochemistry↗

bioRxiv2019

The biosynthetic gene cluster of the C-nucleoside antibiotic pyrazomycin with a rare pyrazole moiety

Pyrazomycin is a rare C-nucleoside antibiotic with a naturally occurring pyrazole ring, whose biosynthetic origin has remained obscure for decades. In this study, we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that StrR-family regulator PyrR is the cluster-situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable-isotope feeding experiments support that PyrN is a new nitrogen-nitrogen bond forming enzyme linking the {varepsilon}-NH2 nitrogen of O_SCPLOWLC_SCPLOW-N6-OH-lysine and -NH2 nitrogen of O_SCPLOWLC_SCPLOW-glutamate. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes constructing the characteristic pyrazole ring.

biochemistry↗