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Abstract: Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX (CoPPIX) cofactor. CoPPIX-containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min–1) while suppressing competing hydrogen evolution pathways. A 2.1 Å crystal structure shows close agreement to the design model with the Co–Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.

Open Access

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Abstract: The role of the cell envelope-associated Rv0132c/FGD2 from Mycobacterium tuberculosis has long been a subject of debate. Importantly, FGD2 is found only in pathogenic mycobacteria, making it a potential drug target. While some suggest it functions as a glucose-6-phosphate dehydrogenase, others propose it acts instead as an F420-dependent hydroxy-mycolic acid dehydrogenase—an activity linked to cell-wall remodeling and inhibition by the anti-tubercular drug pretomanid. Yet, direct evidence for either activity has been lacking. Here, we heterologously express and purify active Mtb-FGD2, and demonstrate that the enzyme binds the F420 cofactor with nanomolar affinity. Crystal structures for both the apo-form and the F420 complex reveal that the Mtb-FGD2 active site architecture is consistent with sugar substrates but notably lacks a phosphate-binding pocket. Biochemical assays confirm that Mtb-FGD2 functions efficiently as an F420-dependent glucose dehydrogenase in vitro. Computational docking combined with molecular dynamics simulations further supports the formation of a catalytically plausible β-D-glucose:F420 ternary complex. When coupled to other F420-dependent enzymes, Mtb-FGD2 readily supports glucose-driven F420.H2-dependent oxidoreductase activity. Our data thus suggest that the Mtb-FGD2 provides reduced F420.H2 in a glucose-dependent manner to support mycobacterial F420.H2-dependent oxidoreductases in the cell envelope.

Open Access

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Abstract: The rapid emergence of RNA therapeutics has highlighted the need for more efficient, scalable and sustainable methods for their manufacture. Biocatalytic approaches hold particular promise, but rely on a secure, sustainable and low-cost supply of nucleoside triphosphate (NTP) building blocks, including those containing chemical modifications. Here we report the development of a biocatalytic approach and engineered enzymes to convert widely available nucleosides into NTPs featuring pharmaceutically relevant modifications using inexpensive phosphate donors. Importantly our strategy obviates the need for ATP as a phosphate donor that complicates NTP isolation using existing methods. To showcase the utility of our approach, we employ an engineered acid phosphatase, polyphosphate kinase and acetate kinase to produce 2′-O-methoxyethyl-ATP (2′-MOE-ATP) and 2′-fluoro-ATP, key building blocks of commercial therapeutics. Finally, we show that crude NTPs from our process can be used directly in enzymatic oligonucleotide synthesis, obviating the need for costly NTP isolation or purification steps.

Open Access

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Abstract: Tuberculosis is the deadliest infectious disease in history and new drugs are urgently required to combat multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Here, we exploit the relience of Mtb on host-derived cholesterol to develop a novel class of antitubercular compounds that target Mtb CYP125 and CYP142; the enzymes that catalyze the first step of cholesterol metabolism. A combination of fragment screening and structure-based drug design was used to identify a hit compound and guide synthetic optimization of a dual CYP125/142 ligand 5m (KD 40–160 nM), which potently inhibits enzyme activity in vitro (KI < 100 nM), and the growth of Mtb in extracellular (MIC99 0.4–1.5 μM) and intracellular assays (IC50 1.7 μM). The structural data and lead compounds reported here will help study Mtb cholesterol metabolism and guide the development of novel antibiotics to combat MDR Mtb.

Open Access

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Abstract: Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter-α-inhibitor (IαI) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC•HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric ‘Half-PTX3’ and a construct missing the 24 N-terminal residues (Δ1-24-PTX3). SAXS determined a length of ∼520 Å for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC•HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC•HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC•HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.