Harding, C. J. | Search bioRxiv

bioRxiv2025

Improved nucleoside (2'-deoxy)ribosyltransferases maximize enzyme promiscuity while maintaining catalytic efficiency

Nucleoside analogues have been extensively used to treat viral and bacterial infections and cancer for the past 60 years. However, their chemical synthesis is complex and often requires multiple steps and a dedicated synthetic route for every new nucleoside to be produced. Wild type nucleoside 2'-deoxyribosyltransferase enzymes are promising for biocatalysis. Guided by the structure of the enzyme from the thermophilic organism Chroococcidiopsis thermalis PCC 7203 (CtNDT) bound to the ribonucleoside analogue Immucillin-H, we designed mutants of CtNDT and the psychrotolerant Bacillus psychrosaccharolyticus (BpNDT) to improve catalytic efficiency with 3'-deoxynucleosides and ribonucleosides, while maintaining nucleobase promiscuity to generate over 100 distinct nucleoside products. Enhanced catalytic efficiency towards ribonucleosides and 3'-deoxyribonucleosides occurred via gains in turnover rate, rather than improved substrate binding. We determined crystal structures of two engineered variants as well as kinetic parameters with different substrates, unveiling molecular details underlying their expanded substrate scope. Our rational approach generated robust enzymes and a roadmap for reaction conditions applicable to a wide variety of substrates. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/641663v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@3ef466org.highwire.dtl.DTLVardef@11f55b0org.highwire.dtl.DTLVardef@32d0dcorg.highwire.dtl.DTLVardef@2e22cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

Cyclic dipeptides are produced by organisms in all domains of life. Many possess biological activities as anticancer and antimicrobial compounds. Oxidations are frequently present in these biologically active peptides. C-C bond oxidation is catalysed by tailoring enzymes, including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their complex three-dimensional arrangement involving multiple copies of two distinct small subunits and unclear mechanistic features underlying substrate selection and catalysis. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), part of the biosynthetic pathway for nocazine natural products. We show NdasCDO forms filaments in solution with a covalently bound FMN cofactor in the interface between three distinct subunits. The enzyme is promiscuous, using many cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH, involving the formation of a radical intermediate. Pre-steady state kinetics, a sizeable solvent kinetic isotope effect and lack of viscosity effects support that a step coupled to FMN regeneration determines the rate of the reaction. Our work dissects the complex mechanistic and structural features of this dehydrogenation reaction. This sets the stage to utilizing NdasCDO as a biocatalyst and expands the FMN-dependent oxidase family to include enzyme filaments.

biochemistry↗