Biocatalysis@TUDelft

During heme biosynthesis in Gram-positive bacteria, coproheme decarboxylase (ChdC) catalyzes the conversion of four-propionate substrate coproheme to the two propionate product heme b. Its active site is universally covered by a flexible linking loop. This study identifies an important histidine residue, which stabilizes the loop in a ChdC homolog. A point mutation increased active site accessibility and subsequently increased oxidative damage caused by cosubstrates. Our results indicate a protective function of the loop.

Coproheme decarboxylase (ChdC) is the terminal enzyme in Gram-positive heme b biosynthesis, an enzyme holding a special importance given its unique structure–function relationship and its necessity for bacterial survival. In the past, the enzyme has been shown to perform a double decarboxylation of two propionate groups on coproheme (Fe(III)-coproporphyrin III), subsequently converting it to heme b (iron-protoporphyrin IX). Notably, the active site of ChdCs is universally covered by a flexible loop. Its importance has not been fully studied but given its position it is assumed to provide steric hinderance for substrates or potential inhibitors to pass. This study aims to investigate its physiological role by introducing a histidine-to-alanine mutation located on the loop of ChdC from Listeria monocytogenes. Molecular dynamics simulation shows an increased flexibility of the structural element in the mutant. As a consequence, various kinetic studies at steady- and presteady-state conditions suggest coproheme binding and active site accessibility are improved. Using simulations and X-ray crystallography, we show evidence that the loop is originally stabilized by a hydrogen bond between S116 and the mutated H117. The higher accessibility also results in a higher susceptibility to damage from oxidative cosubstrates like H₂O₂, suggesting the loop in its wild-type conformation plays a key biological role in regulating the transfer of cosubstrates towards the main substrate coproheme.

Nature Chemical Biology, Published online: 06 May 2026; doi:10.1038/s41589-026-02239-5

Author Correction: Defining and refining the cysteine redoxome through sulfur chemical biology

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced by biosynthetic enzymes that modify genetically encoded precursor peptide backbones and side chains. Genome mining and bioinformatics analyses targeting the multinuclear nonheme iron oxidative (MNIO) enzyme family led to the identification of a Streptomyces thermodiastaticus JCM 4840 RiPP biosynthetic gene cluster, the std cluster, which includes multiple biosynthetic enzymes and a precursor peptide containing a conserved SNKEWQE motif. Using in vitro approaches, we elucidated the modifications installed by the std biosynthetic enzymes. First, a YcaO-TfuA pair thioamidates the backbone of asparagine. Next, a peptidase S8/S53 domain fused to a NodU-like carbamoyltransferase that both carbamoylates the {varepsilon}-amino group of lysine to produce the non-proteinogenic amino acid homocitrulline and cleaves the C-terminal EWQE motif. Finally, a partner protein-MNIO pair bis-hydroxylates the {beta}- and {gamma}-carbon positions of the installed homocitrulline. The formation of homocitrulline and its subsequent modification are unprecedented in RiPP biosynthesis. Moreover, these findings expand the substrate scope of YcaO-TfuA enzymes and MNIOs and identify new roles for carbamoyl transferases in RiPP biosynthesis.

Nature Chemistry, Published online: 06 May 2026; doi:10.1038/s41557-026-02138-1

Optimizing the performance of complex enzymatic reaction networks remains challenging as hidden interactions and unfavourable kinetic barriers often arise when enzymes operate together in networks. Now a model-guided design strategy for generating time-dependent protocols for batch reactions has been developed. This strategy offers a general approach for optimizing multi-step reaction sequences.

The orphan cytochrome P450 CYP107J1 with unknown redox partners was rationally modified into H₂O₂-driven form. The QE (E251Q/T252E) mutant produced valuable chemicals by simply mixing the enzyme with substrate and H₂O₂.

ABSTRACT

Cytochrome P450 monooxygenases (P450s) not only play many physiological roles in oxidative metabolism but are also promising biocatalysts for the synthesis of organic molecules. Bacillus subtilis strain 168, which possesses eight P450 genes, has been extensively studied as a model bacterium, but the catalytic function of CYP107J1 remains to be fully elucidated. Here, we investigated the catalytic function of CYP107J1. Because the genes encoding the reductase components of CYP107J1 could not be identified from the genome sequence, putidaredoxin (Pdx) and its reductase (PdR) were first used to reconstitute the enzymatic activity of CYP107J1. The enzyme showed oxidation activity toward 4-alkylbenzoic acids with a carbon chain length of 3–8, although the activity was low. To enhance the activity of CYP107J1, over that observed when using heterologous redox partners, we next engineered CYP107J1 into a hydrogen peroxide–driven form (i.e., peroxygenase). Only two amino acid substitutions in the active site of CYP107J1 were required for a peroxygenase variant exhibiting 28 times higher catalytic activity toward 4-hexylbenzoic acid than the wild-type enzyme supported by Pdx and PdR. This highly active enzyme enabled detailed characterization of CYP107J1. Interestingly, the engineered enzyme not only oxidized 4-alkylbenzoic acids but also efficiently produced the valuable dye indigo by simply mixing the enzyme with indole and hydrogen peroxide. Enzymes of the CYP107J subfamily are widely distributed among bacteria of genus Bacillus. The findings from this study will facilitate further exploitation of the catalytic potential of CYP107J subfamily enzymes.

We engineered a catalytically enhanced programmable Pyrococcus furiosus Argonaute by integrating directed evolution with structure-guided design, enabling highly sensitive tetracycline detection. This strategy improves the performance of Argonaute-based biosensing and expands its application scope, providing a foundation for field-deployable diagnostics in clinical, environmental, and food safety settings.

In this study, we identify two homologous flavin-dependent enzymes, OxaJ and OtnJ, that catalyze enantiodivergent acyclic peroxide formation in oxanthromicin biosynthesis. We show that a conserved structural loop functions as a redox gate, restricting NADPH access and thereby favoring peroxide formation, whereas removal of this loop restores NADPH engagement and switches the reaction outcome to hydroxylation.

ABSTRACT

Peroxy natural products, including endoperoxides, acyclic peroxides, and hydroperoxides, are widely distributed across all domains of life, with many, such as artemisinin and prostaglandins, serving as clinically important agents. However, the enzymatic mechanisms by which nature installs O─O bonds have largely remained elusive. To date, only a limited number of endoperoxide-forming enzymes have been identified, while the enzymatic basis for acyclic peroxide assembly remains unknown. Here, we identify two homologous flavin-dependent enzymes, OxaJ and OtnJ, that catalyze enantioselective acyclic peroxide formation in the biosynthesis of oxanthromicin natural products. A conserved structural motif acts as a redox gate by blocking NADPH access to the active site, thereby promoting peroxide installation. Removal of this motif permits NADPH binding and redirects the enzyme's activity toward hydroxylation. This work establishes the first example of peroxide formation by a flavin-dependent enzyme and introduces redox gating as a previously unrecognized strategy for controlling oxidative divergence in enzymatic catalysis.

Nature Chemistry, Published online: 04 May 2026; doi:10.1038/s41557-026-02125-6

Promiscuous interactions underpin natural protein evolution, but ways to harness such promiscuity to design new functions remain underexplored. Now it is shown that mapping this promiscuity with geometric precision in a de novo protein can guide its redesign into a fluorophore binder and an efficient enzyme approaching the diffusion limit.

The catalytic substrates of TET, ALKBH, and KDM proteins span the entire central dogma. This study investigated the substrate recognition diversification driven by the structural evolution of their DSBH domains and identified the pro-DSBH, revealing that it creates conditions for protein substrate binding.

Ten-eleven translocation (TET), AlkB homolog (ALKBH), and histone lysine demethylase (KDM) proteins belong to the 2-oxoglutarate (2OG) and ferrous iron-dependent oxygenases, which catalyze substrates spanning the entire central dogma (DNA–RNA–Protein). It serves as a model for understanding how functional diversity is shaped by structural changes within the central dogma. The evolutionary characteristics of the core catalytic domain double-stranded β-helix (DSBH) are the main reasons for their specific substrate recognition ability. Nonetheless, the structural biological explanations remain ambiguous. Here, we constructed the sequence evolutionary tree of the full-length and DSBH catalytic domains from seven prevalent mammalian species. The DSBH domain evolutionary trajectory was generated by multispecies structure fitting and mapping. Taking humans as a reference, the relationships among the three subfamilies, KDM, ALKBH, and TET, were depicted. In conjunction with the evolutionary tree and domain map, this method analyzed the emergence and extinction of α-helix and β-sheet structures and length variations in pivotal regions to ascertain the structural history of DSBH domains across subfamilies. Furthermore, reduced amino acid analysis and prospective mapping were utilized to investigate the correlation between the structural progression and functional assessment of the DSBH structure. A new perspective has arisen, highlighting variations in the proregion of the DSBH domain as essential for its particular substrate recognition function, as this domain determines the binding conditions for protein substrates. This provides a new angle for understanding the evolution of proteins from simple structures to complex functions and leads to new advances for the bioengineering field.

Phospholipid N-methyltransferases (Pmts) synthesize phosphatidylcholine in many bacteria. In this study, we compared the catalytic mechanisms of two bacterial Pmt classes: the Rhodobacter (R-) and the Sinorhizobium (S-) type. Representative enzymes for each class were derived from Rubellimicrobium thermophilum and Agrobacterium tumefaciens, respectively. Our results indicate that tyrosine (Y) residues play a critical role in the catalytic mechanisms of both enzymes, although their modes of activation differ.

Phosphatidylcholine (PC), the predominant phospholipid in eukaryotic membranes, also plays a crucial role in certain bacterial species, often mediating interactions with eukaryotic hosts. In bacteria, a major pathway for PC biosynthesis involves the three-step methylation of phosphatidylethanolamine, catalyzed by phospholipid N-methyltransferases (Pmts). While the binding site for the methyl donor S-adenosyl- l -methionine is well characterized in Pmt enzymes, the detailed mechanism of methyl group transfer remains poorly understood. In this study, we combined computational and biochemical approaches to identify the amino acid residues critical for the catalytic activity of two distinct Pmt classes: the Rhodobacter (R)-type enzyme from Rubellimicrobium thermophilum (RtPmtA) and the Sinorhizobium (S)-type enzyme from Agrobacterium tumefaciens (AtPmtA). Despite low sequence identity, both enzyme types share similar reaction mechanisms, with tyrosine residues playing key roles in methyl group transfer. In RtPmtA, two highly conserved tyrosines located within the substrate-binding pocket on the N-terminal αA-helix are critical for enzymatic function. In contrast, AtPmtA depends on a single tyrosine buried in the protein core for catalysis. These findings reveal distinct active site architectures and suggest that R-type and S-type enzymes have evolved class-specific structural strategies for tyrosine activation. This divergence highlights the evolutionary flexibility of Pmt enzymes, despite their shared catalytic function.

The radical S-adenosylmethionine (SAM) superfamily comprises more than 800,000 enzymes that use [Fe4S4] clusters to initiate radical chemistry that mediates an exceptionally broad range of chemical transformations. Within this superfamily, cobalamin (Cbl)-dependent radical SAM enzymes constitute a major subclass predominantly associated with methylation reactions. However, several notable members catalyze non-methylase reactions, for which the mechanistic role of Cbl is poorly understood. Bacteriochlorophyll biosynthesis enzyme BchE is a Cbl-dependent radical SAM enzyme that catalyzes a six-electron oxidation of Mg-protoporphyrin IX monomethylester (MPE) to protochlorophyllide (PChlide), installing a ketone and forming the fifth ring of bacteriochlorophyll under anaerobic conditions. Although prior in vivo and in vitro studies have demonstrated a requirement for Cbl, SAM, and a low-potential reductant, detailed mechanistic analysis has been impeded by the inability to obtain soluble, catalytically active enzyme. Here, we report the successful isolation and spectroscopic characterization of BchE, enabling the first in vitro reconstitution of its enzymatic activity. Using both chemical and biological reducing systems, we observe the formation of PChlide along with proposed reaction intermediates and several off-pathway products. These results provide new insight into the oxidative chemistry mediated by Cbl in non-methylase radical SAM enzymes and establish BchE as a tractable model for elucidating how cobalamin is deployed in this understudied subclass.