Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

Abstract

CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast‐carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form‐II and form‐II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s−1—sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.

Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off

“…This result is also compatible with the mechanistic models of RuBisCO (Farquhar, 1979), and is supported by the recent discovery of enzyme variants which exhibit the highest kcatC ever recorded at the expense of poor CO2 affinities (i.e. KC values >250 µM) (Davidi et al, 2020). Nevertheless, the dependency between CO2 affinity and turnover, despite being the strongest pairwise correlation between kinetic traits observed, is still only partial (~30 % variance explained), and is substantially attenuated relative to the coefficients conventionally cited from studies investigating limited numbers of species (Tcherkez, Farquhar and Andrews, 2006;Savir et al, 2010).…”

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli

“…For kinetic measurements of diverse rubiscos, see recent meta-analyses by Flamholz et al, 2019 ; Iñiguez et al, 2020 . For recent measurements of the R. eutropha , R. rubrum , and S. elongatus enzymes, see Davidi et al, 2020 ; Occhialini et al, 2016 ; Satagopan and Tabita, 2016 . ( B ) Growth rate and yield of CCMB1:p1A+vec depend on the CO 2 concentration, with higher CO 2 improving growth (‘vec’ denotes pFA-sfGFP).…”

“…Net carboxylation was calculated as the carboxylation rate less ½ the oxygenation rate, which presumes a plant-type photorespiratory pathway that loses one CO 2 for every two oxygenation reactions. ( B ) Bacterial Form II rubiscos are typically found in organisms living in low O 2 environments and, accordingly, display low CO 2 specificities (S C/O ≈ 10) and relatively high maximum carboxylation rates (k cat,C ≈10–20 s −1 , Davidi et al, 2020 ). As such, Form II rubiscos do not perform well in ambient CO 2 and O 2 concentrations.…”

Quantifying the taxonomic bias in enzymology

“…This was illustrated in dramatic fashion when a single study reported 1,003 reference genomes selected solely because they maximized coverage of phylogenetic space, and which collectively increased the number of known protein sequence families by over 10% 11 . Sequence similarity networks and genome mining algorithms are also being used increasingly to identify enzymes with divergent sequences that expand our understanding, have biotechnological value, or both 13,28–32 . For example, a recent study identified 33,000 sequences for the carbon‐fixing enzyme, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO), in genomic and metagenomic datasets 28 .…”