Biology subjects

Hochberg, G. K. A.

Publications and source records attributed to Hochberg, G. K. A..

10 recordsLinked to original sources

bioRxiv2026

Interface swapping orchestrates carbon transfer in the archaeal acetyl-CoA decarbonylase/synthase

The Wood-Ljungdahl pathway is one of biologys most ancient routes for carbon fixation and energy metabolism, used by organisms such as methanogenic archaea. One of its central metabolic complexes is the acetyl-CoA decarbonylase/synthase (ACDS) complex, catalyzing acetyl-CoA synthesis and cleavage through the co-ordinated action of carbon monoxide dehydrogenase (CODH), acetyl-CoA synthase (ACS), and corrinoid iron-sulfur protein (CoFeSP). Unlike bacterial CODH/ACS, archaeal ACDS lacks a stable bifunctional CODH-ACS architecture, raising the question of how reactive CO and methyl intermediates are efficiently transferred between catalytic modules. Using cryo-electron microscopy, crosslinking mass spectrometry, small-angle X-ray scattering, and biophysical analyses, we resolved the organization and dynamics of the [~]2 MDa archaeal ACDS supercomplex from Methanosarcina acetivorans. We identified CoFeSP as a central architectural scaffold that self-assembles into hexa- to octameric oligomers via a conserved N-terminal region of the CdhD subunit. This scaffold likely tethers CODH and ACS through conserved disordered terminal regions, positioning the catalytic modules in the complexs periphery. We propose a mechanism in which ACS transiently alternates between CODH and CoFeSP, enabling efficient CO and methyl-group transfer without stable binary complexes. This dynamic organization represents a fundamental difference to the stable bifunctional CODH/ACS in bacteria, highlighting how transient interactions enable efficient acetyl-CoA metabolism in archaea.

biochemistry↗

bioRxiv2026

Coordination of sequential RNase activities in an ancient molecular machine

The evolutionary mechanisms by which molecular machines incorporate catalytic subunits and coordinate their functions are still poorly understood. Here, we study the molecular evolution of the RNA exosome, an essential RNA-processing machine built from a 9-subunit core (Exo9). Human and yeast Exo9 are catalytically inactive and serve as a recruiting hub for the peripheral RNase Rrp44. The core of modern exosomes descends from an active RNase in Archaea. Using ancestral sequence reconstruction, biochemical and structural characterization, we illuminate how Exo9 evolved from an enzyme to a regulatory hub. The ancestral Exo9 was an active, distributive RNase that already cooperated with Rrp44. Cryo-electron microscopy reveals how RNA-binding modulates the conformation of Exo9, thereby promoting allosteric recruitment of Rrp44. Exo9 begins substrate trimming before the RNA slips past its active site. The RNA can thereby be handed over to Rrp44 to be processed further, which rationalizes the coordination of consecutive RNase activities. We hypothesize that the same allosteric pathway still exists in the human exosome, implying that this mechanism persisted for over a billion years. Our work thus illuminates the evolutionary tinkering that produced a complex molecular machine and a central component of the eukaryotic cell.

evolutionary biology↗

bioRxiv2024

Methylthio-alkane reductases use nitrogenase metalloclusters for carbon-sulfur bond cleavage

Methylthio-alkane reductases convert methylated sulfur compounds to methanethiol and small hydrocarbons, a process with important environmental and biotechnological implications. These enzymes are classified as nitrogenase-like enzymes, despite lacking the ability to convert dinitrogen to ammonia, raising fundamental questions about the factors controlling their activity and specificity. Here, we present the first molecular structure of the methylthio-alkane reductase, which reveals large metalloclusters, including the P-cluster and the [Fe8S9C]-cluster, previously only found in nitrogenases. Our findings suggest that distinct metallocluster coordination, surroundings, and substrate channels, determine the activity of these related metalloenzymes. This study provides new insights into nitrogen fixation, sulfur-compound reduction, and hydrocarbon production. We also shed light on the evolutionary history of P-cluster and [Fe8S9C]-cluster-containing reductases emerging prior to nitrogenases.

biochemistry↗

bioRxiv2024

Bottom-up reconstruction of minimal pyrenoids provides insights into the evolution and mechanisms of carbon concentration by EPYC1 proteins

Membraneless organelles play essential roles in cellular processes. In various photosynthetic organisms, they confer carbon concentrating mechanisms (CCMs). One example is the pyrenoid in Chlamydomonas reinhardtii, a liquid-phase separated organelle that localizes and improves carbon fixation via the intrinsically-disordered protein (IDP) EPYC1. Modern-day pyrenoids are complex structures, which makes it impossible to study the function of EPYC1, especially whether EPYC1 alone confers carbon concentration, and how EPYC1 could have initiated the evolution of pyrenoids. Here, we developed a bottom-up approach to study the function of EPYC1 and its sequence-function space across evolution. We demonstrate that modern-day EPYC1-sequences, but not other IDPs, induce liquid-phase separation of Rubisco into minimal pyrenoids with functional CCMs. Using ancestral sequence reconstruction, we trace the evolution of pyrenoids and demonstrate that selection acted on carboxylation rate, and in selected cases on specificity, providing unexpected perspectives on the design and function of natural and synthetic pyrenoids.

biochemistry↗

bioRxiv2024

Layered entrenchment maintains essentiality in protein-protein interactions.

Protein complexes composed of strictly essential subunits are abundant in nature and arise through the gradual complexification of ancestral precursor proteins followed by their co-evolution with the newly recruited components. Essentiality arises during co-evolution by the accumulation of changes that are tolerated in the complex state but would be deleterious for the standalone complex components. While this theoretical framework to explain how essentiality arises has been proposed long ago, it is unclear which factors cause essentiality to persist over evolutionary timescales. In this work we show that the central enzyme of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), rapidly started to depend on a newly recruited interaction partner through multiple, genetically distinct mechanisms that affect stability, solubility, and catalysis. We further demonstrate that layering multiple mechanisms of essentiality can lead to the persistence of essentiality, even if any given mechanism reverts through chance or selection. More broadly, our work highlights that new interaction partners can drastically re-shape which substitutions are tolerated in the proteins they are recruited into. This can lead to the rapid evolution of multi-layered essentiality through the exploration of areas of sequence space that are only accessible in the complex state.

biochemistry↗

bioRxiv2023

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and, hence, are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterised the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterised by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double-deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of {Delta}fdxC and {Delta}fdxN strains, in conjunction with differing plasmid complementation behaviours of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation. ImportanceNitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. Production of ammonia by diazotrophic organisms, harbouring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, though we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterised the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centres. Additionally, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviours.

biochemistry↗

bioRxiv2022

Molecular basis and design principles of a system for switchable front-rear polarity and directional migration

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions vary. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that inhibits the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.

microbiology↗

bioRxiv2022

Evolutionary Inspired Engineering of Megasynthetases

Many clinically used drugs are derived from or inspired by bacterial natural products that often are biosynthesised via non-ribosomal peptide synthetases (NRPS), giant megasynthases that activate and join individual amino acids in an assembly line fashion. Since NRPS are not limited to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would allow the biotechnological generation of complex peptides including linear, cyclic and further modified natural product analogues, e.g. to optimise natural product leads. Here we describe a detailed phylogenetic analysis of several bacterial NRPS that led to the identification of a new recombination breakpoint within the thiolation (T) domain that is important for natural NRPS evolution. From this, an evolution-inspired eXchange Unit between T domains (XUT) approach was developed which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

synthetic biology↗

bioRxiv2021

Green light perception paved the way for the diversification of GAF domain photoreceptors

Photoreceptors are proteins that sense incident light and then trigger downstream signaling events. Phytochromes are linear tetrapyrrole-binding photoreceptors present in plants, algae, fungi, and various bacteria. Most phytochromes respond to red and far-red light signals. Among the phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show much more diverse optical properties covering the entire visible region. Both phytochromes and cyanobacteriochromes share the GAF domain scaffold to cradle the chromophore as the light-sensing region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here we utilize ancestral sequence reconstruction and report that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore enables the photoreceptor to perceive green light. The ancestral cyanobacteriochromes show modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense green-rich irradiance rather than red light, which is preferentially utilized for photosynthesis. In contrast to plants and green algae, many cyanobacteria can utilize green light for photosynthesis with their special light-harvesting complexes, phycobilisomes. The evolution of green/red sensing cyanobacteriochromes may therefore have allowed ancient cyanobacteria to acclimate to different light environments by rearranging the absorption capacity of the cyanobacterial antenna complex by chromatic acclimation. Significance StatementLight serves as a crucial environmental stimulus affecting the physiology of organisms across all kingdoms of life. Photoreceptors serve as important players of light responses, absorbing light and actuating biological processes. Among a plethora of photoreceptors, cyanobacteriochromes arguably have the wealthiest palette of color sensing, largely contributing to the success of cyanobacteria in various illuminated habitats. Our ancestral sequence reconstruction and the analysis of the resurrected ancestral proteins suggest that the very first cyanobacteriochrome most probably responded to the incident green-to-red light ratio, in contrast to modern red/far-red absorbing plant phytochromes. The deprotonation of the light-absorbing pigment for green light-sensing was a crucial molecular event for the invention of the new class of photoreceptors with their huge color tuning capacity.

biochemistry↗

bioRxiv2018

It takes a dimer to tango: Oligomeric small heat-shock proteins dissociate to capture substrate

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and their mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show that substructural dimers of plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and non-dimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced had wild-type-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for activity. In addition, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different than the Ta16.9 x-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) showed greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Size and secondary structure changes with temperature revealed that folded sHsp dimers interact with substrate, and support dimer stability as a determinant of chaperone efficiency. These data yield a model in which sHsp dimers capture substrate prior to assembly into larger, heterogeneous sHSP-substrate complexes for subsequent substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

molecular biology↗