Biology subjects

Lewandowski, J. R.

Publications and source records attributed to Lewandowski, J. R..

5 recordsLinked to original sources

bioRxiv2025

Substrate scope and catalytic mechanism of α, β-epoxyketone synthase EpnF illuminated by in-situ esterase-mediated deprotection

, {beta}-Epoxyketones are an important class of bacterial natural products with wide-ranging potential applications in oncology, immunology, and infectious disease. Their clinical potential derives from the , {beta}-epoxyketone pharmacophore, which covalently modifies the N-terminal catalytic threonine residue of proteasome {beta}-subunits with high selectivity. Although a synthetic , {beta}-epoxyketone (Carfilzomib) is approved for clinical use, stereo-controlled synthesis of the pharmacophore remains challenging, involving either multiple steps and energy-intensive processes, or unsustainable reagents. Unusual flavoenzymes catalyze the assembly of the pharmacophore in , {beta}-epoxyketone biosynthesis. A detailed understanding of the substrate scope and catalytic mechanism of these enzymes has thus far been limited by the intrinsic instability of their - (di)methyl-{beta}-ketoacid substrates. Here, we report the development and application of an esterase-mediated unmasking strategy for in-situ generation of these substrates from the corresponding methyl esters. Using this approach, we demonstrate that EpnF, the epoxyketone synthase involved in eponemycin / TMC-86A biosynthesis, tolerates a broad range of synthetic substrate analogs, including several with N-terminal protecting groups widely used in peptide synthesis. These findings establish that EpnF has the potential to be developed into a useful biocatalyst for the chemoenzymatic synthesis of dipeptidyl epoxyketone precursors of clinically approved drugs and drug candidates. To elucidate the molecular basis for catalysis of , {beta}-epoxyketone formation by EpnF, substrate docking and molecular dynamics simulations were performed on a well-validated AlphaFold model, providing support for a previously proposed decarboxylation-dehydrogenation-monooxygenation mechanism. Site-directed mutagenesis and LC-MS analysis validated the proposed roles of key active-site residues in substrate positioning and catalysis of epoxide formation. Collectively, these results demonstrate that EpnF and related enzymes belong to a new class of internal flavoprotein monooxygenases and provide a foundation for developing epoxyketone synthases into useful biocatalysts for the sustainable synthesis of high-value ,{beta}-epoxyketones.

biochemistry↗

bioRxiv2025

Molecular characterisation of the acyltransferase-acyl carrier protein interface in a fungal highly reducing polyketide synthase.

Iterative polyketide synthases (iPKSs) rely on communication between acyl carrier protein (ACP) and acyltransferase (AT) domains to ensure efficient delivery of starter and extender substrates during biosynthesis. However, the molecular determinants governing the AT:ACP interface remain poorly understood. Here, we use the fungal highly reducing PKS, SimG, a component of the cyclosporin biosynthetic pathway, as a model system to dissect the AT:ACP interface. Using alanine scanning mutagenesis combined with a high-throughput intact-protein mass spectrometry assay, we identified epitope-forming residues that affect AT:ACP interaction. These experimental constraints were used to guide docking and molecular dynamics simulations to produce a data-driven structural model of the SimG AT:ACP complex in a catalytically competent geometry. We also demonstrate that the SimG AT domain transacylates ACP domains from a range of fungal PKS architectural classes, highlighting significant interface plasticity. These insights advance our fundamental understanding of domain communication in these enigmatic megasynthases and provide a foundation for rational engineering to expand substrate scope towards novel polyketide scaffolds.

biochemistry↗

bioRxiv2025

Molecular basis for depsipeptide HDAC inhibitor combinatorial biosynthesis

Polyketides and nonribosomal peptides are important natural product classes with wide-ranging medical and agricultural applications. The analogous enzymatic logic employed by bacterial modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) enables the assembly of hybrid products. One important group of polyketide-nonribosomal peptide hybrids is exemplified by the HDAC-targeting drug romidepsin. This group is assembled by combinatorial biosynthesis involving fusion of a conserved Zn2+-binding pharmacophore to a variable peptide-based cap. Here, we use gene proximity searching to identify the FR-901375 biosynthetic gene cluster in Pseudomonas chlororaphis subsp. piscium DSM 21509. Comparison of the PKS-NRPS encoded by this gene cluster with those assembling related depsipeptide HDAC inhibitors suggests a novel subunit docking modality enables interaction between the conserved pharmacophore and variable cap biosynthetic machineries. This hypothesis was validated using crosstalk assays, mutagenesis, AlphaFold predictions, and carbene footprinting, providing new insight into the evolution of mechanisms for hybrid polyketide-nonribosomal peptide combinatorial biosynthesis.

biochemistry↗

bioRxiv2024

Molecular chaperone BiP controls activity of the ER stress sensor Ire1 through interactions with its oligomers

The complex multistep activation cascade of Ire1 involves changes in the Ire1 conformation and oligomeric state. Ire1 activation enhances ER folding capacity, in part by overexpressing the ER Hsp70 molecular chaperone BiP; in turn, BiP provides tight negative control of Ire1 activation. This study demonstrates that BiP regulates Ire1 activation through a direct interaction with Ire1 oligomers. Particularly, we demonstrated that the binding of Ire1 luminal domain (LD) to unfolded protein substrates not only trigger conformational changes in Ire1-LD that favour the formation of Ire1-LD oligomers but also exposes BiP binding motifs, enabling the molecular chaperone BiP to directly bind to Ire1-LD in an ATP-dependent manner. These transient interactions between BiP and two short motifs in the disordered region of Ire1-LD are reminiscent of interactions between clathrin and another Hsp70, cytoplasmic Hsc70. BiP binding to substrate-bound Ire1-LD oligomers enables unfolded protein substrates and BiP to synergistically and dynamically control Ire1-LD oligomerisation, helping to return Ire1 to its deactivated state when an ER stress response is no longer required.

biophysics↗

bioRxiv2023

Molecular basis for short-chain thioester hydrolysis by acyl hydrolase domains in trans-acyltransferase polyketide synthases

Polyketide synthases (PKSs) are multi-domain enzymatic assembly lines that biosynthesise a wide selection of bioactive natural products from simple building blocks. In contrast to their cis-acyltransferase (AT) counterparts, trans-AT PKSs rely on stand-alone AT domains to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. Trans-AT PKS gene clusters also encode acyl hydrolase (AH) domains, which are predicted to share the overall fold of AT domains, but hydrolyse aberrant acyl chains from ACP domains, thus ensuring efficient polyketide biosynthesis. How such domains specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH domain and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AH and ACP domains contributes to recognition of cognate and non-cognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the production of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by trans-AT PKSs, and provide foundations for future bioengineering efforts.

biochemistry↗