Inter-subunit coupling enables fast CO₂-fixation by reductive carboxylases

Abstract

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...

Infrared Spectroscopy Reveals Metal independent Carbonic Anhydrase Activity in Crotonyl CoA Carboxylase/Reductase

“…To rationalize the observed inhibition of CA-like activity through substrate binding, we performed atomistic MD simulations in the presence of CO2 using the X-ray crystal structure of KsCcr with the NADPH cofactor and side product B-CoA (ternary complex, PDB ID 6NA4). [26] Note that the enzyme is a tetramer that shows half-site reactivity, i.e., exists as dimer of open and closed subunits (colored orange and green in Notably, active site residues H365 and E171, which we associate with CA-like activity, adopted different geometries in the open and closed conformations. Compared to the open subunit, the distance between E171 and H365 was 3 Å shorter in the closed subunit, and the conserved water molecule µW was hydrogen-bonded between the two residues (Figure 5).…”

“…For comparison we also studied the X-ray structure without substrate (binary complex, PDB ID 6NA4) that presents similar geometries of both residues in the closed and open subunits. [26] We presume that H365 can act as base in its neutral, monoprotonated state, initiating proton abstraction from µW, and thus forming the hydroxyl ion for subsequent CO2 hydration. To determine the protonation state of H365 in different subunits of the binary and ternary complexes, we calculated its pKa shifts (Table S1).…”

“…The absence of NADPH or NADP + is not expected to affect CO2 binding or H365 protonation because the coenzyme does not bind directly to the substrate binding site. [26] In contrast, the presence of C-CoA or B-CoA in the ternary complex increases the pKa of the putative proton acceptor H365, thereby eliminating its ability to activate µW water by proton transfer, and simultaneously diminishes CO2 binding. This can explain the experimentally observed reduction of CA-like activity and is in line with the fact that the active enzyme ternary complex promotes CO2 fixation [26] , and not CO2 hydration.…”