Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO 2 -fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open-and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO 2 -fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter-and intrasubunit communications of this remarkably efficient CO 2 -fixing enzyme during catalysis.

Developing new carbon dioxide (CO 2 ) fixing enzymes is a prerequisite to create new biocatalysts for diverse applications in chemistry, biotechnology and synthetic biology. Here we used bioinformatics to identify a “sleeping carboxylase function” in the superfamily of medium-chain dehydrogenases/reductases (MDR), i.e. enzymes that possess a low carboxylation side activity next to their original enzyme reaction. We show that propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus possess carboxylation yields of 3 ± 1 and 4.5 ± 0.9%. We use rational design to engineer these enzymes further into carboxylases by increasing interactions of the proteins with CO 2 and suppressing diffusion of water to the active site. The engineered carboxylases show improved CO 2 -binding and kinetic parameters comparable to naturally existing CO 2 -fixing enzymes. Our results provide a strategy to develop novel CO 2 -fixing enzymes and shed light on the emergence of natural carboxylases during evolution.

Reduced nitrogen species are key nutrients for biological productivity in the oceans. Ammonium is often present in low and growth-limiting concentrations, albeit peaks occur during collapse of algal blooms or via input from deep sea upwelling and riverine inflow. Autotrophic phytoplankton exploit ammonium peaks by storing nitrogen intracellularly. In contrast, the strategy of heterotrophic bacterioplankton to acquire ammonium is less well understood. This study revealed the marine bacterium Phaeobacter inhibens DSM 17395, a Roseobacter group member, to have already depleted the external ammonium when only ∼⅓ of the ultimately attained biomass is formed. This was paralleled by a three-fold increase in cellular nitrogen levels and rapid buildup of various nitrogen-containing intracellular metabolites (and enzymes for their biosynthesis) and biopolymers (DNA, RNA and proteins). Moreover, nitrogen-rich cells secreted potential RTX proteins and the antibiotic tropodithietic acid, perhaps to competitively secure pulses of external ammonium and to protect themselves from predation. This complex response may ensure growing cells and their descendants exclusive provision with internal nitrogen stocks. This nutritional strategy appears prevalent also in other roseobacters from distant geographical provenances and could provide a new perspective on the distribution of reduced nitrogen in marine environments, i.e. temporary accumulation in bacterioplankton cells.

19Enoyl-CoA carboxylases/reductases (ECRs) are the most efficient CO2-fixing enzymes described 20 to date, outcompeting RubisCO, the key enzyme in photosynthesis in catalytic activity by more 21 than an order of magnitude. However, the molecular mechanisms underlying ECR's 22 extraordinary catalytic activity remain elusive. Here we used different crystallographic 23 approaches, including ambient temperature X-ray Free Electron Laser (XFEL) experiments, to 24 study the dynamic structural organization of the ECR from Kitasatospora setae. K. setae ECR is 25 a homotetramer that differentiates into a dimer of dimers of open-and closed-form subunits 26 in the catalytically active state, suggesting that the enzyme operates with "half-site reactivity" 27 to achieve high catalytic rates. Using structure-based mutagenesis, we show that catalysis is 28 synchronized in K. setae ECR across the pair of dimers by conformational coupling of catalytic 29 domains and within individual dimers by shared substrate binding sites. Our results provide 30 unprecedented insights into the dynamic organization and synchronized inter-and intra-31 subunit communications of nature's most efficient CO2-fixing enzyme during catalysis. 32The capture and conversion of atmospheric CO2 remains a challenging task for chemistry, resulting in an 34 ever-increasing interest to understand and exploit CO2 fixation mechanisms offered by biology 1 . The 35recently described family of enoyl-CoA carboxylases/reductases (ECRs) represent the most efficient CO2-36 fixing enzymes found in nature to date 2,3 . ECRs catalyze the reductive carboxylation of a variety of enoyl- 37CoA thioester substrates at catalytic rates that are up to 20-fold higher than Ribulose-1,5-bisphosphate 38 carboxylase/oxygenase (RubisCO), an enzyme involved in the first carbon fixation step in the Calvin-39 Benson cycle of photosynthesis 1,4 .40 ECRs catalyze the reduction of α,β-unsaturated enoyl-CoAs using the reduced form of the cofactor 41 nicotinamide adenine dinucleotide phosphate (NADPH). This generates a reactive enolate species, which 42 acts as a nucleophile to attack a CO2 molecule 2,3,5 . The structural details of the carboxylation reaction have 43 remained elusive, due in part to the lack of high-resolution structures of ECRs containing catalytic 44 intermediates and carboxylated products. Currently, there are five available ECR structures. However, 45 2 they all have different substrate specificities, ranging from short-(PDB: 3HZZ, 3KRT) to long-chain (4A0S 6 ) 46 and aromatic enoyl-CoA substrates (4Y0K 7 ), and are from different biological backgrounds including 47 primary (i.e. central carbon) metabolism (PDB: 4GI2) and secondary metabolism. Moreover, most of them 48 were co-crystalized with NADPH or NADP+ only and do not contain CO2, enoyl-CoA substrates or acyl-CoA 49 products. This significantly limits our structural understanding of the enzyme's catalytic mechanism. 50The aim of this study was to provide a detailed structural understanding of the carboxylation reaction...