Biology subjects

Prywes, N.

Publications and source records attributed to Prywes, N..

5 recordsLinked to original sources

bioRxiv2025

Diversity-driven biochemical survey reveals dimeric structural origin of rubisco

Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural diversity of rubisco, we systematically sampled and synthesized a library of diverse rubisco sequences with an emphasis on clades that have previously not been characterized. Our updated phylogenetic analysis reveals that many deep-branching rubiscos assemble as dimers, supporting a dimeric origin for the superfamily -- in contrast to the ecologically dominant hexadecameric form I. Additionally, we discover and structurally characterize the largest rubisco described to date, originating from a cryptic, early-branching subclade with novel structural folds that have previously not been observed in the rubisco superfamily. By integrating biochemical data with an updated phylogenetic framework, we propose a revised nomenclature for the rubisco protein family that reflects current insights and will better accommodate future discoveries.

biochemistry↗

bioRxiv2024

Cyanobacteria from marine oxygen deficient zones encode both form I and form II rubiscos

Cyanobacteria are highly abundant in the marine photic zone and primary drivers of the conversion of inorganic carbon to biomass. To date, all studied Cyanobacterial lineages encode carbon fixation machinery hinged upon form I rubisco enzymes within a CO2-concentrating carboxysome. Here, we report that the AMZ IB lineage of Prochlorococcus from global oxygen deficient zones (ODZs) harbor both form I and form II rubisco enzymes, the latter of which are typically non-carboxysomal and possess biochemical properties tuned towards low oxygen environments. Our analyses reveal that these cyanobacterial form II enzymes are functional in vitro and were likely acquired via lateral gene transfer from proteobacteria. Global metagenomic read recruitment demonstrates that Prochlorococcus with form II rubisco are essentially restricted to ODZs in the Eastern Tropical Pacific, suggesting that acquisition may confer an advantage specifically under low-O2 conditions. Populations of AMZ IB Prochlorococcus express both forms of rubisco in situ, with the highest form II rubisco expression at depths where both oxygen and light are particularly low, possibly as a mechanism to increase the efficiency of photoautotrophy under energy limitation. Our findings expand the diversity of carbon fixation configurations in the microbial world and may have implications for the overall capacity of ODZs to sequester carbon.

microbiology↗

bioRxiv2023

A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases

Using directed evolution, engineered aminoacyl-tRNA synthetases (aaRS) have been developed that enable co-translational incorporation of numerous noncanonical amino acids (ncAAs) into proteins in living cells. Until now, the selection of such novel aaRS mutants has relied on coupling their activity to the expression of a reporter protein with a selectable phenotype. However, such translation-dependent selection schemes are incompatible with exotic monomers that diverge structurally from canonical -amino acids and are suboptimal substrates for the ribosome. To enable the ribosomal incorporation of such exotic monomers, a two-step solution is needed: A) Engineering an aaRS to acylate its cognate tRNA with the exotic monomer, without relying on ribosomal translation as a readout, and B) Subsequent engineering of the ribosome to accept the resulting acylated tRNA for translation. Here, we report a platform for aaRS engineering that directly selects for tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the associated barcode-containing tRNAs from an oxidative treatment designed to damage the 3'-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the M. alvus pyrrolysyl-tRNA synthetase from a naive library that charge noncanonical amino acids.

synthetic biology↗

bioRxiv2023

Mapping the biochemical landscape of rubisco

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

biochemistry↗

bioRxiv2018

Revisiting tradeoffs in Rubisco kinetic parameters

Rubisco is the most abundant enzyme in the biosphere and one of the best-characterized enzymes. Based on correlations between Rubisco kinetic parameters, it is widely posited that tradeoffs embedded in the catalytic mechanism constrain its specificity and maximum catalytic rate. However, the reasoning that established this view was based on data from {approx}20 organisms. We re-examine these tradeoff models using a dataset from {approx}300 organisms. Most correlations are substantially attenuated, with the inverse relationship between carboxylation kcat and specificity SC/O being a key example. Only one tradeoff model survives in our dataset. In this model, increasing catalytic efficiency (kcat/KM) for carboxylation requires increased catalytic efficiency for the competing oxygenation reaction, evidenced by strong power-law correlation between catalytic efficiencies. Our results imply that Rubisco evolution is constrained primarily by the physicochemical limits of O2/CO2 discrimination, which should reframe efforts to engineer this very central enzyme.

systems biology↗