Biocatalysis@TUDelft

Triazolopyridines are an important class of heterocycles in the pharmaceutical industry and materials sciences. In particular, [1,2,3]triazolo[1,5a]pyridines have emerged as stable and versatile diazo compound precursors for performing carbene-mediated transformations. Despite their wide range of applications in chemical synthesis, their preparation is often reliant on oxidative cyclization methods using stoichiometric oxidants in organic solvent, limiting their application in chemoenzymatic synthesis. We have recently discovered that vanadium-dependent haloperoxidase (VHPO) enzymes are effective catalysts for performing the oxidative cyclization of 2-pyridyl ketone hydrazones to give [1,2,3]triazolo[1,5a]pyridines through cryptic diazo formation. Herein, we have developed a chemoenzymatic protocol for conversion of 2-pyridyl ketones to [1,2,3]triazolo[1,5a]pyridines in a single vessel through the in situ generation of 2-pyridyl ketone hydrazones followed by VHPO-catalyzed oxidative cyclization to give [1,2,3]triazolo[1,5a]pyridines in high yield and chemoselectivity.

The plant Aster tataricus and its endophytic fungus Cyanodermella asteris are sources of a unique class of halogenated pentapeptides called astins. In addition, the fungus Talaromyces islandicus produces the astin-related class of pentapeptides, cyclochlorotines. Through molecular networking and mass spectrometry fragmentation data, we uncover a huge chemical diversity among the astin and cyclochlorotine pool of pentapeptides, which also reflects the genetic homology of both nonribosomal peptide synthetase (NRPS) systems and deepens our understanding of astin biosynthesis and enzymology.

ABSTRACT

The plant Aster tataricus L.f. is known as the main source of astin-type cyclopentapeptides. However, recent studies have shown that the endophytic fungus Cyanodermella asteris contains a biosynthetic gene cluster (BGC) ast encoding for astin core-structure biosynthesis and that it produces three astins, namely astins C, F, and G. The foodborne fungus Talaromyces islandicus produces the structurally highly related cyclochlorotine mycotoxins encoded by the BGC cct with significant genetic homology to ast from C. asteris. We thus became interested in exploring and comparing the astin-type peptide diversity found in these three source organisms to answer the long-standing question of the true metabolic origin of the astins. Mass spectrometry-based molecular networking and fragmentation trees revealed that A. tataricus, C. asteris, and T. islandicus indeed contain many more derivatives, hence uncovering a huge peptide chemodiversity resulting from promiscuous NRPS systems regarding amino acid selectivity. Both NRPS systems produce structurally related and even a number of identical compounds. Our findings reveal comprehensive metabolic insights strongly indicating that the endophyte C. asteris is the sole producer of the astin peptide family and, contrary to previous assumptions, metabolic cross-talk with the host plant is unlikely to have a major impact on astin structural diversity.

Structural homology between DPOR and nitrogenase enables insertion of the L-cluster from NifEN into Pchlide-free BchNB to create a hybrid that reduces N₂ and C₁ substrates, revealing that BchNB naturally hosts an L-cluster binding site adjacent to the Pchlide pocket while offering insight into the shared evolution and engineering potential of these homologous enzyme systems.

The dark-operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key step in photosynthetic pigment biosynthesis. Structurally and mechanistically related to nitrogenase, DPOR consists of a reductase (BchL) and a catalytic component (BchNB) homologous to the reductase (NifH) and catalytic component (NifDK) of Mo-nitrogenase. Structural alignment of Rhodobacter capsulatus (Rc) BchNB with Azotobacter vinelandii (Av) NifDK and the cofactor maturase NifEN reveals a conserved α₂β₂ architecture and a shared cofactor-insertion path linking their respective prosthetic-like group/cofactors (Pchlide, M-cluster, L-cluster), suggesting the possibility of generating chimeric proteins with novel reactivities. Herein, Pchlide-free RcBchNB (RcBchNB^apo) is reconstituted with the L-cluster extracted from AvNifEN to yield a hybrid protein (RcBchNB^L) capable of reducing N₂ and C₁ substrates (CN⁻, CO) to NH₃ and hydrocarbons, respectively, in the presence of a strong reductant (Eu^II-DTPA). In contrast, reconstituting Pchlide-bound RcBchNB with the L-cluster yields minimal activity, indicating that Pchlide and the L-cluster compete for a common binding site, as supported by Boltz-2 modeling. These findings support the hypothesis of an intertwined evolution of photosynthetic and nitrogen-fixing enzymes and outline a framework for engineering chimeric metalloenzymes that couple light capture with nitrogenase-like catalysis in the future.

Selective modification of peptides and proteins is vital for their development as therapeutics. Here, the adenylation domain of a carboxylic acid reductase enzyme from Segniliparus rugosus (CARsr-A) was used to generate reactive acyl adenylates in situ from carboxylic acids. Functionalized and simple fatty acids were incorporated with excellent selectivity at the N-termini of therapeutically relevant peptides and proteins.

Abstract

The selective modification of proteins and peptides is an important chemical biology tool with a wide variety of applications, including the production of biopharmaceuticals or the study of post-translational modifications. In particular, the selective acylation of the N-terminus over side chains in peptides and proteins is a highly desirable but challenging reaction in this field. Current methods have a range of shortcomings, including lack of selectivity or narrow substrate scope. Here we report a biomimetic approach using the in situ enzymatic reagent activation (ERA) of carboxylic acids with ATP to generate acyl-adenosine monophosphates. This method displays high selectivity for the N-termini of peptides and proteins, including pharmaceutically relevant liraglutide, glucagon and insulin. The ERA acylation tolerates a broad range of unsubstituted and substituted fatty acids, including azido and dicarboxylic acids, thus making it suitable for N-terminal bioorthogonal labelling strategies. Moreover, this strategy can also be applied to the modification of antibodies. In general, the ERA acylation is a versatile and bioorthogonal method that we envisage finding wider applications in the field of bioconjugation and the production of stable peptide and protein conjugates.

TM1131 from Thermotoga maritima was identified as a novel branched-chain and aromatic amino acid aminotransferase. TM1040, previously identified as a histidinol-phosphate aminotransferase, was also characterized. These enzymes are involved in the final step of the biosynthetic pathways of branched-chain and aromatic amino acids in T. maritima.

The hyperthermophile Thermotoga maritima does not possess a typical branched-chain amino acid aminotransferase or aromatic amino acid aminotransferase, leaving the biosynthetic pathways of these amino acids unclear. In this study, we identified and characterized a novel branched-chain and aromatic amino acid aminotransferase (TM1131). We also characterized a histidinol-phosphate aminotransferase (TM1040) with reported aminotransferase activity toward aromatic amino acids. TM1131 exhibited broad substrate specificity and the highest activity toward branched-chain and aromatic amino acids as an amino donor and toward corresponding 2-oxoacids as an amino acceptor. TM1040 also showed broad substrate specificity, with the highest activity toward l-lysine and l-arginine as an amino donor, and toward 2-oxoacids corresponding to l-methionine, l-leucine, and l-phenylalanine. Additionally, we investigated the multifunctionality of these two enzymes to explore other potential amino acid metabolic activities. Intriguingly, TM1131 displayed aspartate 4-decarboxylase activity, albeit with lower catalytic efficiency than measured for aminotransferase activity. TM1131 is involved in the final step of the biosynthetic pathways of branched-chain and aromatic amino acids, to which TM1040 also likely contributes.

A chemoenzymatic total synthesis of the H. pylori SS1 O-antigen octadecasaccharide was achieved by stereoconvergent [6 + 4] assembly and protecting-group-controlled enzymatic site-specific fucosylation. Glycan microarray screening of the synthetic O-antigen and its fragments identified a simpler antigenic epitope, providing a key target for glycoconjugate vaccine design.

Abstract

Helicobacter pylori infection represents a major global health challenge, characterized by high prevalence, significant association with gastric cancer, and rising antibiotic resistance. Carbohydrate-based vaccines targeting the O-antigen of lipopolysaccharide (LPS) present a promising alternative to conventional antimicrobial therapies. To explore the immunogenicity of LPS O-antigen from clinical isolate H. pylori SS1, we report an integrated chemoenzymatic strategy for the first synthesis of its octadecasaccharide O-antigen and related fragments for antigenicity evaluation. Our strategy features modular chemical synthesis of a decasaccharide precursor containing a high-carbon sugar (D,D-Hep) residue, a unique oligomeric β1,2-linked ribofuranosyl tetrasaccharide motif and a switchable glucosamine (GlcNH₂) residue through stereoconvergent [6 + 4] assembly, followed by protecting-group-controlled enzymatic elongation to precisely install hybrid Lewis antigen moiety (Le ^y -Le ^x ) in a site-specific fucosylation manner to afford the target octadecasaccharide bearing five challenging 1,2-cis-glycosidic linkages. Chemical stereoselective construction of 1,2-cis-glucosidic and 1,2-cis-fucosidic linkages was accomplished by reagent-controlled glycosylation and 4-O-acyl remote participation, respectively. Enzymatic site-specific installation of the remaining three 1,2-cis-fucosidic linkages was achieved using two robust fucosyltransferases and a strategically designed GlcNH₂ residue. Glycan microarray-based screening of the synthetic O-antigen and its subunits with H. pylori-infected patient sera identified an undecasaccharide as a simpler and key epitope for vaccine development.

Electrochemical activation of galactose oxidase (GalOx) has emerged as a sustainable alternative to peroxide-driven systems for aerobic alcohol oxidation. Here, we present a systematic investigation of wild-type GalOx under electrochemical conditions for the oxidation of non-carbohydrate primary alcohols. While benzylic substrates undergo selective enzyme-mediated oxidation to the corresponding alde-hydes, we identify a prominent competing pathway in which weakly bound substrates are directly oxidized at the electrode surface, fre-quently resulting in over-oxidation to carboxylic acids. Using 4-methoxybenzyl alcohol as a diagnostic probe, we disentangle the enzymat-ic and non-enzymatic pathways and demonstrate that both applied potential and electrode material exert decisive control over product selectivity. In contrast, aryl 1,2-diols are oxidized exclusively through a direct anodic mechanism, completely bypassing enzymatic turno-ver. Collectively, these results define critical operational boundaries for electro-enzymatic GalOx catalysis and underscore the necessity of rigorous enzyme-free controls in the development of selective electrochemical biocatalytic oxidation platforms.

Paclitaxel, a clinically potent anticancer drug derived from Taxus species, faces persistent challenges in sustainable supply. Synthetic biology presents substantial opportunities for its de novo production, particularly with recent breakthroughs in elucidating its intricate biosynthetic pathways. However, its heterologous biosynthesis is significantly constrained by key bottlenecks, including pathway complexity, poor P450 expression, and inefficient metabolic flux. In this study, we explore how synthetic biology facilitates pathway decoding and reconstruction and propose strategies involving nonclassical chassis such as plant-associated cyanobacteria and filamentous fungi to enhance P450 compatibility. We also present a pragmatic framework for the rational application of state-of-the-art tools, including cell-free systems, synthetic microbial consortia, hybrid chemoenzymatic synthesis, and machine learning, to sustainably produce paclitaxel and other natural products.

De novo protein design has not yet achieved the creation of proteases capable of selectively cleaving any desired peptide bond within a native protein with high precision. Here, we report the use of the flow-based generative model Proteus2 to design metalloproteases by generating enzyme-substrate complexes conditioned on a target sequence and a predefined catalytic motif. Our approach employs a two-step encapsulation strategy to create clamp-like metalloproteases that bind the target peptide in a manner that maximizes substrate sequence specificity. This generative process simultaneously optimizes the precise positioning of the target peptide bond in a catalytically competent configuration and accurately scaffolds the transition state catalytic residues--both essential for specific and efficient catalysis. Using this strategy, we designed zinc metalloproteases targeting three distinct cleavage sites within the aggregation-prone regions of amyloid-{beta} (A{beta}), a key pathogenic factor in Alzheimers disease. Experimental characterization validated five enzymes, each capable of precise cleavage at the intended sites with high specificity and minimal or undetectable activity on non-cognate substrates. On average, these enzymes accelerated peptide bond hydrolysis by more than 107-fold relative to the uncatalyzed reaction. Together, these results demonstrate the potential of sequence-guided, generative approaches for developing programmable, sequence-specific proteolysis and lay the groundwork for future applications in basic research and therapeutic development.

MHETases are enzymes implicated in polyethylene terephthalate (PET) biodegradation. The present study elucidates the structural determinants that result in increased mono(2-hydroxyethyl) terephthalate (MHET) degradation by a feruloyl esterase, which has been engineered to resemble MHETase active site. The crystal structures of the variant in apo and benzoic acid bound state reveal the changes induced by the introduced mutation, specifically the formation of a hydrogen bond and a trans to cis isomerization of a peptide bond in the vicinity of the catalytic site. Molecular dynamics simulations demonstrate the stabilization of the loop harboring the engineered residue, as well as an expansion of the substrate binding cleft, which would facilitate accommodation of a broader variety of substrates, indicative of a promiscuous biocatalyst. EnzymeEnzyme Commission Number: EC 3.1.1.73; Uniprot accession code: A0A1D3S5H0_FUSOX

The axial ligand of heme is a key determinant of reactivity in heme-dependent enzymes, yet its systematic engineering remains challenging due to the limited chemical diversity of natural amino acids. Here, we demonstrate that replacing the native histidine axial ligand with a non-natural analog provides an effective strategy to regulate heme enzyme activity. By site-specifically incorporating δVin-H into APEX2, we reprogrammed the electronic structure of the heme center and unlocked catalytic activity toward aromatic amine substrates. Directed evolution of the APEX2-VinH enzyme yielded APEX3-VinH, which exhibits a pronounced enhancement in catalytic efficiency and robustness for aromatic amine oxidation. Notably, optimization of the δVin-H synthetase enabled highly efficient incorporation. This work establishes axial ligand engineering via genetic code expansion as a general and powerful strategy to reprogram the catalytic repertoire of heme enzymes, with broad implications for biocatalysis and the development of next-generation proximality labeling tools.

Computational modeling of enzymes provides molecular-level insight into catalysis, but the preparation of quantum mechanical (QM) calculations starting from experimental structures is a significant bottleneck for high-throughput studies. Automated tools developed to accelerate this process may fail to generalize across distinct active site chemistries and geometries. To overcome these limitations, we present QuantumPDB, a Python package that automates the generation of hierarchical coordination/interaction spheres around an active center to create QM cluster models directly from raw protein structures. The workflow integrates structure cleaning, protonation state assignment, and QM calculation setup. It uses chemically meaningful models constructed from contact-based interaction spheres derived from Voronoi tessellation, enabling accurate representation of complex active site geometries. We provide an overview of our modular code and describe how it may be employed to automate high-throughput protein screening. To demonstrate its utility, we curated a dataset of 989 holo-enzymes from the PDB and performed QM calculations on 1,673 enzyme cluster models of 842 of these enzymes. Analysis of computed properties suggests that enzyme environments simulated with density functional theory consistently modulate substrate charge toward neutrality and reduce the substrate dipole moment. This phenomenon appears to be general, even in cases where the active site consists predominantly of neutral residues. By automating and standardizing multi-sphere QM model construction, QuantumPDB provides a robust platform for large-scale, data-driven investigations of proteins.

Nonheme Fe enzyme isopenicillin N synthase was reprogrammed and evolved as an efficient nitrogen migratase IPNS_Nim, converting diverse azanyl esters to valuable l-arylglycines with up to 16 000 TTN and 97:3 e.r. IPNS_Nim and ACCO_Nim allowed enantiodivergent preparation of both l- and d-arylglycines. Mechanistic studies revealed a change in the rate-determining step and H atom transfer enantioselectivity for these nonheme enzymes.

Abstract

We describe the reprogramming and directed evolution of nonheme Fe enzyme isopenicillin N synthase (IPNS) as an efficient biocatalyst for 1,3-nitrogen migration reactions via an unnatural mechanism. Directed evolution of isopenicillin N synthase from Emericella nidulans furnished a quadruple mutant (EniIPNS V185L I187V S102I R279H, IPNS_Nim), enabling the conversion of a range of azanyl esters into N-protected l-arylglycines. IPNS_Nim achieved a TTN of 16 000 and a TOF of 1200 min⁻¹. This TTN surpassed state-of-the-art small-molecule Fe catalysts by 330-fold and represented the highest TTN value reported for a nonheme Fe enzyme in a new-to-nature reaction. IPNS_Nim and our previously evolved ACCO_Nim (ACCO: 1-aminocyclopropane-1-carboxylic acid oxidase) exhibited complementary enantiopreference, allowing enantioselective synthesis of either l- or d-arylglycines—essential building blocks in clinically important peptide therapeutics. Mechanistic studies revealed a biocatalyst-controlled switch in the rate-determining step (RDS): While the hydrogen atom transfer (HAT) step is the RDS for ACCO_Nim-catalyzed nitrogen migration, it is likely not with IPNS_Nim. Moreover, while ACCO_Nim exhibits almost no enantioselectivity in this HAT step, IPNS_Nim confers excellent enantiocontrol over HAT. Computational studies using density functional theory calculations and molecular dynamics simulations suggested that IPNS and ACCO adopt two different substrate binding modes. Classical MD simulations shed light on important interactions between the substrate and active-site residues that control the substrate binding mode and enantioselectivity.