Biology subjects

Hitchcock, A.

Publications and source records attributed to Hitchcock, A..

11 recordsLinked to original sources

bioRxiv2026

Assembly and activation of the AAA+ motor of the multi-subunit cobaltochelatase

Cobalamin is a complex yet essential cobalt-containing cofactor synthesized exclusively by prokaryotes and archaea. Aerobic cobalamin biosynthesis requires the CobNST multi-subunit class I cobaltochelatase for cobalt incorporation. Cobalt chelation is dependent on the energy transfer from the CobST AAA+/VWA motor unit to the substrate-binding catalytic unit (CobN). However, the mechanism for assembly and function of CobNST cobaltochelatase remains poorly understood. Here, we used cryo-EM to solve the structures of the AAA+ motor CobS, in the absence and presence of ATP and its VWA-partner protein CobT. Our data reveal that the AAA+ CobS assembles into various conformations, including hexameric planar rings and different spiral left-handed assemblies: hexamers, double rings and filaments. In the absence of nucleotide, the CobT N-terminal domain interacts with CobS left-handed oligomers, remaining in a half-assembled, nonfunctional motor unit. In the CobST complex, ATP induces conformational rearrangement of CobS from left-handed helical oligomers to a right-handed closed hexamer. The simultaneous repositioning of the CobT VWA domain to the AAA+ central pore shifts the motor unit to a functional state, ready-to-drive CobN-catalyzed chelation reaction. These findings define the mechanism for assembly and activation of the class I cobaltochelatase CobST motor.

biochemistry↗

bioRxiv2026

Outer membrane 'PusCD' complexes coordinate phospho(inositol)lipid utilisation in plant Bacteroidota

Gram-negative bacteria belonging to the phylum Bacteroidota are prominent and ecologically important members of host-associated microbiomes. A feature of Bacteroidota species is the presence of diverse outer membrane TonB-dependent transporter and cognate surface exposed lipoprotein (TBDT-SLP) complexes. Many 'SusCD-like' TBDT-SLPs coordinate complex carbohydrate capture from the environment. Here, we show the model soil Bacteroidota species Flavobacterium johnsoniae synthesises two TBDT-SLP complexes (named PusCD1 and PusCD2) that are essential for growth on phosphatidylinositol, a member of an abundant lipid class within plant plasma membranes, as a phosphorus source. Structural characterisation of ligand-bound PusCD1 complex from F. johnsoniae membranes reveals a new binding mechanism for organophosphorus and broadens the known functional repertoire of this class of transporters. Diverse PusCD-like complexes are widespread among plant associated Bacteroidota, pointing to a major, previously unrecognised route for the recycling of organophosphorus compounds in the rhizosphere, with potential implications for engineering microbial-mediated phosphorus recovery from organic waste streams.

microbiology↗

bioRxiv2026

A 13-subunit c-ring in the Chlamydomonas chloroplast ATP synthase lowers the H⁺/ATP cost of carbon fixation

The chloroplast F1Fo ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H+/ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO2 fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 [A] structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c14 in a chloroplast, and with a lower predicted H+/ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching {gamma}-subunit abolishes the contact with the catalytic {beta}-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H+/ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

biochemistry↗

bioRxiv2026

Structure of chlorophyll synthase in complex with the LHC-like protein HliD

Chlorophyll is the central cofactor in oxygenic photosynthesis, responsible for both light capture in antenna complexes and light-driven charge separation in photosystems. The tetraprenyl tail of chlorophyll is attached to the chlorophyllide macrocycle by the transmembrane enzyme chlorophyll synthase (ChlG). In the cyanobacterium Synechocystis sp. PCC 6803, ChlG forms a complex with the LHC-like high-light-inducible protein HliD. To understand the substrate specificity and catalytic mechanism of ChlG, and how it is photoprotected by HliD, we determined the structure of a ChlG2HliD2 complex in both substrate-free and geranylgeranyl pyrophosphate (GGPP)-bound states using cryogenic electron microscopy (cryo-EM). A homodimer of HliD, which binds four chlorophylls and two zeaxanthin molecules in a quenched state, is flanked by two ChlG monomers. AlphaFold modelling placed chlorophyllide adjacent to the structurally resolved GGPP bound to the active site of ChlG. Cryo-EM data, site-directed mutagenesis and molecular dynamics were used to formulate a molecular mechanism for ChlG catalysis. The structure of the ChlG2HliD2 complex shows how Hlips bind to the synthase, reveals the arrangements of carotenoids and chlorophylls that mediate energy dissipation, and sheds light on the evolution of eukaryotic LHC antennae from their cyanobacterial ancestors.

plant biology↗

bioRxiv2025

The impact of carotenoid energy levels on the exciton dynamics and singlet-triplet annihilation in the bacterial Light-Harvesting 2 complex

The light-harvesting 2 (LH2) complex of purple phototrophic bacteria plays a critical role in absorbing solar energy and distributing excitation energy. Exciton dynamics within LH2 complexes are controlled by the structural arrangement and energy levels of the bacteriochlorophyll (BChl) and carotenoid (Car) pigments. However, there is still debate over the competing light-harvesting versus energy-dissipation pathways. In this work, we compared five variants of the LH2 complex from genetically modified strains of Rhodobacter sphaeroides, all containing the same BChls but different Cars with increasing conjugation: zeta-carotene (N=7; LH2Zeta), neurosporene (N=9; LH2Neu), spheroidene (N=10; LH2Spher), lycopene (N=11; LH2Lyco), and spirilloxanthin (N=13; LH2Spir). Absorption measurements confirmed that Car excited state energy decreased with increasing conjugation. Similarly, fluorescence spectra showed that the B850 BChl emission peak had an increasing red shift from LH2Zeta[->](LH2Neu/LH2Spher)[->]LH2Lyco[->]LH2Spir. In contrast, time-resolved fluorescence and ultrafast transient absorption (fs-TA) revealed similar excited state lifetimes ([~]1 ns) for all complexes except LH2Spir ([~]0.7 ns). From fs-TA analysis, an additional [~]7 ps non-radiative dissipation step from B850 BChl was observed for LH2Zeta. Further, singlet-singlet and singlet-triplet annihilation studies showed a [~]50% average fluorescence lifetime reduction in LH2Zeta at high laser power and high repetition rate, compared to [~]10-15% reductions in LH2Neu/LH2Spher/LH2Lyco and minimal lifetime change in LH2Spir. In LH2Zeta, the fastest decay component (<50 ps) became prominent at high repetition rates, consistent with strong singlet-triplet annihilation. Nanosecond TA measurements revealed long-lived (>40 s) BChl triplet states in LH2Zeta and signs of damage caused by singlet oxygen, whereas other LH2s showed faster triplet quenching ([~]18 ns) by Cars. These findings highlight a key design principle of LH2 complexes: the Car triplet energy must be significantly lower than the BChl triplet energy to efficiently quench BChl triplets that otherwise act as potent "trap states" causing exciton annihilation in laser-based experiments or photo-damage in native membranes.

biophysics↗

bioRxiv2024

Tethering ferredoxin-NADP+ reductase to photosystem I promotes photosynthetic cyclic electron transfer

Fixing CO2 via photosynthesis requires ATP and NADPH. Linear electron transfer (LET) supplies both metabolites, yet depending on environmental conditions, additional ATP is required which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is set remains largely unknown. Ferredoxin(FD)-NADP+ reductase (FNR) has been suggested to act as the switch, channelling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or CET when bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. We circumvented this problem by using CRISPR-Cas9 gene editing in Chlamydomonas reinhardtii to create a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth relative to the wild type, and decreased LET and non-photochemical quenching (NPQ) under high CO2 conditions. However, higher rates of CET and increased NPQ were found in the mutants under low CO2 or low O2 conditions where CET is normally up-regulated. Thus, rather than PSI-FNR interactions promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.

biochemistry↗

bioRxiv2024

Hybrid xyloglucan utilisation loci are prevalent among plant-associated Bacteroidota

The plant hemicellulose xyloglucan (XyG) is secreted from the roots of numerous plant species, including cereals, and contributes towards soil aggregate formation in terrestrial systems. Whether XyG represents a key nutrient for plant-associated bacteria is unclear. The phylum Bacteroidota are abundant in the plant microbiome and provide several beneficial functions for their host. However, the metabolic and genomic traits underpinning their success remain poorly understood. Here, using proteomics, bacterial genetics, and genomics, we revealed that plant-associated Flavobacterium, a genus within the Bacteroidota, can efficiently utilise XyG through the occurrence of a distinct and conserved gene cluster, referred to as the Xyloglucan Utilisation Loci (XyGUL). Flavobacterium XyGUL is a hybrid of the molecular machinery found in gut Bacteroides spp., Cellvibrio japonicus, and the plant pathogen Xanthomonas. Combining protein biochemistry, computational modelling and phylogenetics, we identified a mutation in the enzyme required for initiating hydrolysis of the XyG polysaccharide, an outer membrane endoxyloglucanase glycoside hydrolase family 5 subfamily 4 (GH5_4), which enhances activity towards XyG. A subclade of GH5_4 homologs carrying this mutation were the dominant form found in soil and plant metagenomes due to their occurrence in Bacteroidota and Proteobacteria. However, only in members of the Bacteroidota spp., particularly Flavobacterium spp. was such a remarkable degree of XyGUL conservation detected. We propose this mechanism enables plant-associated Flavobacterium to specialise in competitive acquisition of XyG exudates and that this hemicellulose may represent an important nutrient source, enabling them to thrive in the plant microbiome, which is typified by intense competition for low molecular weight carbon exudates.

microbiology↗

bioRxiv2024

Sulfoquinovosyl diacylglycerol is required for dimerization of the Rhodobacter sphaeroides RC-LH1 core complex

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer in which 14 LH1 subunits form a crescent-shaped antenna around one RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding two RCs. The PufX polypeptide augments the five RC and LH subunits, and in addition to providing an interface for dimerization, PufX also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form a quinone channel; protein-Z, of unknown function but which is unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginines. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

biochemistry↗

bioRxiv2023

Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom

Although AlphaFold2 (AF2) and RoseTTAFold (RF) have transformed structural biology by enabling high-accuracy protein structure modeling, they are unable to model covalent modifications or interactions with small molecules and other non-protein molecules that can play key roles in biological function. Here, we describe RoseTTAFold All-Atom (RFAA), a deep network capable of modeling full biological assemblies containing proteins, nucleic acids, small molecules, metals, and covalent modifications given the sequences of the polymers and the atomic bonded geometry of the small molecules and covalent modifications. Following training on structures of full biological assemblies in the Protein Data Bank (PDB), RFAA has comparable protein structure prediction accuracy to AF2, excellent performance in CAMEO for flexible backbone small molecule docking, and reasonable prediction accuracy for protein covalent modifications and assemblies of proteins with multiple nucleic acid chains and small molecules which, to our knowledge, no existing method can model simultaneously. By fine-tuning on diffusive denoising tasks, we develop RFdiffusion All-Atom (RFdiffusionAA), which generates binding pockets by directly building protein structures around small molecules and other non-protein molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we design and experimentally validate proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and optically active bilin molecules with potential for expanding the range of wavelengths captured by photosynthesis. We anticipate that RFAA and RFdiffusionAA will be widely useful for modeling and designing complex biomolecular systems.

biochemistry↗

bioRxiv2021

A highly active phosphate-insensitive phosphatase is widely distributed in nature

The regeneration of bioavailable phosphate from immobilised organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three major phosphatases, known as PhoA, PhoX and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these three phosphatase families is negatively regulated by phosphate availability and thus these enzymes play a major role in scavenging phosphorus only during times of phosphate scarcity. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the remineralisation of phosphate in the environment. Using Flavobacterium johnsoniae as the model, we reveal PafA is highly active towards phosphomonoesters. Unlike other major phosphatases, PafA is fully functional in the presence of its metabolic product, phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. PafA, which is constitutively produced under all growth conditions tested, rapidly remineralises phosphomonoesters producing significant quantities of bioavailable phosphate that can cross feed into neighbouring cells. pafA is both abundant and highly expressed in the global ocean and abundant in plant rhizospheres, highlighting a new and important enzyme in the global phosphorus cycle with applied implications for agriculture as well as biogeochemical cycling. We speculate PafA expands the metabolic niche of Bacteroidetes by enabling utilisation of abundant organophosphorus substrates in the presence of excess phosphate, when other microbes are rendered incapable. Significance statementPhosphorus is an essential element for all life on Earth. Global primary production, and thus the ability for oceans and soils to drawdown atmospheric carbon dioxide, is in part controlled by the availability of inorganic phosphate. Likewise, global food production is also reliant on adequate supplies of phosphorus to both plants and animals. A major fraction of the total phosphorus pool exists as organic phosphorus, which requires mineralisation to phosphate prior to incorporation into cellular biomolecules. This important process is performed by enzymes known as phosphatases. Here, we reveal that the unique bacterial phosphatase, PafA, is a key player in the global phosphorus cycle and presents a major route for the regeneration of bioavailable phosphate required for both primary and secondary production.

microbiology↗

bioRxiv2017

A rhamnose-inducible system for precise and temporal control of gene expression in cyanobacteria

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

synthetic biology↗