Aug 2026· Journal of the American Chemical Society· 0 citations· 41 references
TL;DR
This systematic analysis of ATP-dependent diazotases from actinomycetes that catalyze the condensation of nitrite with aromatic amines establishes group 3 diazotases as promising, engineerable biocatalysts for selective and efficient diazo installation.
Abstract
The diazo group is a highly valuable functional group in organic synthesis; however, its conventional preparation requires harsh conditions. Here, we provide a systematic analysis of ATP-dependent diazotases from actinomycetes that catalyze the condensation of nitrite with aromatic amines. Sequence similarity network analysis of AMP-binding enzymes revealed two families (type Ia/Ib), with type Ib further classified into 14 groups. In vitro assays confirmed the diazotization activities of the diazotases from 7 groups in the type Ib family, each displaying distinct substrate preferences. Remarkably, diazotases in group 3 exhibited broad substrate specificity and superior catalytic efficiency. The dimeric structure of a representative enzyme, Mco01_40450, was determined at 3.08 Å resolution by cryogenic electron microscopy with C2 symmetry. Subsequent symmetry expansion and 3D classification revealed the structure with AMP, pyrophosphate, and 4-aminohydrocinnamic acid bound to the active site at a resolution of 3.16 Å. Expansion of the substrate-binding pocket and its entrance via amino acid substitution enabled the reaction of bulkier aromatic amines. Unlike classical chemical diazotization, Mco01_40450 operates under mild aqueous conditions and is compatible with sensitive protecting groups. Our findings highlight the structural and functional diversity of diazotases and establish group 3 diazotases as promising, engineerable biocatalysts for selective and efficient diazo installation.
Enzyme engineering serves as a powerful tool in biocatalysis, enabling the development of enzymes with improved stability, activity, and specificity for a range of academic, industrial, and pharmaceutical applications. However, a limited understanding of sequence–structure–function relationships in terpene synthases, the enzymes that form the complex polycyclic hydrocarbon skeletons of terpenoid natural products, presents a major challenge in predicting and engineering the products of terpene synthases. In this study, we investigated the product profiles of two bacterial variediene synthases, OdVS from Olivibacter domesticus and PsVS from Prauserella shujinwangii, and found that they share some products with the bacterial phomopsene synthase, PmS from Allokutzneria albata, despite low sequence similarity. Thirteen diterpenes were isolated and structurally elucidated including two previously unreported compounds. A series of variants of OdVS, PsVS, and PmS were constructed by targeting conserved residues around the active site and aided in the identification of key residues that control the cyclization pathway. Ultimately, mutation of a single residue, PsVSY86L, was found to switch the major product of PsVS from the tricyclic variediene to the tetracyclic phomopsene, although this switch came at the cost of significantly reduced overall yield indicating a tradeoff between activity and product diversification.
It is indicated that combining A-domain substrate prediction with genome mining is an effective strategy for discovering NRPs in Aspergillus, expanding the understanding of their biosynthetic potential.
Hua-Wei Lv, Zhi-Yan Zhang, Jia-Gui Tang et al.· Archives of Microbiology· 0 citations
[NiFe] hydrogenases reversibly catalyze hydrogen oxidation and proton reduction at a Ni–Fe active site coordinated by four cysteine (Cys) residues in two CXXC motifs within the catalytic large subunit. A subset of these enzymes contains selenocysteine (Sec) in place of Cys in the C-terminal CXXC motif, forming [NiFeSe] hydrogenases with enhanced oxygen tolerance and hydrogen production activity. However, such Sec-containing variants have been reported only in Groups 1 and 3 [NiFe] hydrogenases. Here, we present a comprehensive dataset of Group 4 [NiFeSe] hydrogenases based on a genomic survey combined with UGA stop-codon read-through analysis. The dataset comprises 47 non-redundant protein sequences from nine bacterial phyla. Sec substitutions were exclusively identified in the N-terminal CXXC motif, including 27 CXXU-, 4 UXXC-, and 16 UXXU-type sequences, indicating the emergence of non-canonical Sec-containing motifs in Group 4. Phylogenetic analysis revealed a sporadic distribution across three distinct lineages, indicating at least three independent origins of Sec-containing variants from Cys-type ancestors. Such substitutions were found across diverse ecosystems. Genomic context analysis further suggests that these Sec-containing enzymes form energy-converting complexes, similar to those formed by other Group 4 enzymes. The Sec-containing variants co-occur with Sec biosynthesis genes and a conserved guanine residue in the apical loop of Sec insertion sequence elements. These findings provide new insights into the evolution of Sec utilization in [NiFe] hydrogenases.
Masamitsu Takano, Masao Inoue, Riku Aono et al.· Frontiers in Microbiology· 0 citations
Isobutene is an important platform chemical that is still predominantly produced from petroleum-derived feedstocks. The ferulic acid decarboxylase (Fdc) catalyzed decarboxylation of 3-methylcrotonic acid provides a green and sustainable route to bio-based isobutene. However, the poor activity of native Fdc toward this non-natural substrate severely limits its practical application. Here, we developed an integrated enzyme-engineering strategy that combines sequence-cluster mining with distal-site engineering to improve catalytic performance. The engineered triple-site mutant V132C/Y298F/S484A (3MUT) exhibited 5.3-fold the isobutene production of WT. Coupling this mutant with reaction-process optimization and cofactor engineering increased the isobutene yield to 67.9%, representing the highest reported yield to date. Mechanistic studies revealed that distal-site mutations in 3MUT reshaped the active site by disrupting the M293-mediated hydrogen bond that blocks the substrate to active site, then further promoting favorable hydrogen-bond interactions with the substrate via R183 and E292, respectively. These findings establish an efficient route for sustainable bio-based isobutene production and demonstrate the potential of distal-site engineering for improving UbiD-family decarboxylases.
Ting Feng, Xuanyu Cao, Li-Ran Yang et al.· Bioresource Technology· 0 citations
The oxidative modification of complex natural products is critical for their biological activity. However, the enzymatic mechanisms that facilitate three-step, six-electron transfer reactions, such as the conversion of a methyl group to a carboxylic acid, on cyclopeptide scaffolds remain poorly understood. This study characterizes IlaL, a cytochrome P450 monooxygenase from Streptomyces atratus, which catalyzes a six‑electron methyl‑to‑carboxylic acid conversion during the biosynthesis of the potent antimycobacterial cyclopeptide ilamycins. IlaL possesses a noncanonical “Asp-Asn (DN)” dioxygen activation dyad and an extended, flexible BC-loop, which together enable sequential three-step oxidation. The high-resolution co-crystal structure of IlaL bound to the substrate ilamycin B2 reveals an enlarged cavity and dynamic gating conferring precise regioselectivity and broad substrate tolerance. Biochemical and phylogenetic analyses confirm the essential role of the DN dyad and show that DN‑type cytochrome P450 monooxygenases occupy a distinct, recently emerged phylogenetic clade. These findings provide structural and mechanistic insights into six‑electron oxidative modification and establish design principles for versatile biocatalysts enabling late‑stage natural product diversification.
Ming Peng, Qiao-Ling Wu, Ying-Ying Chen et al.· ACS Catalysis· 0 citations
Orcinol synthase (RdORS) from Rhododendron dauricum is a plant type III polyketide synthase involved in the biosynthesis of orsellinic acid-derived metabolites. In contrast to the related Cannabis sativa tetraketide synthase (CsTKS), which preferentially accepts medium-chain acyl-CoAs, RdORS selectively utilizes short-chain starter substrates. Here, we investigated the structural basis underlying this substrate selectivity by combining X-ray crystallography, mutational analysis, and biochemical characterization. The crystal structure of RdORS revealed that its catalytic cavity is substantially smaller than that of CsTKS because of a bulky tryptophan (Trp) 357 residue positioned at the cavity bottom. In vitro enzymatic assays demonstrated that wild-type RdORS efficiently generated tetraketide-derived products from acetyl- and butyryl-CoAs with three malonyl-CoAs, whereas productive tetraketide formation progressively diminished as starter-substrate chain length increased. Structural analysis of the RdORS Trp357S mutant revealed marked cavity expansion without perturbation of the overall catalytic framework. Correspondingly, the Trp357S substitution enabled RdORS to utilize medium-chain acyl-CoAs up to decanoyl-CoA, thereby partially recapitulating the substrate preference of CsTKS. Thus, our results provided direct structural evidence that Trp357 is a key structural determinant underlying the distinct starter-substrate preferences of RdORS.
Y. Nakashima, S. Y. Y. Hnin, Subin Kim et al.· Chemical and pharmaceutical...· 0 citations
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