skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Four amino acids define the CO 2 binding pocket of enoyl-CoA carboxylases/reductases
Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2within the active site of Ecr fromKitasatospora setae. Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2fixation. Altogether, our study reveals unprecedented molecular details of selective CO2binding and C–C-bond formation during the catalytic cycle of nature’s most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2in biology and chemistry.  more » « less
Award ID(s):
1231306
PAR ID:
10588788
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ;
Publisher / Repository:
Proceedings of the National Academy of Sciences
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
116
Issue:
28
ISSN:
0027-8424
Page Range / eLocation ID:
13964 to 13969
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Angewandte Chemie International Edition)
    Abstract Carbon‐supported nitrogen‐coordinated single‐metal site catalysts (i.e., M−N−C, M: Fe, Co, or Ni) are active for the electrochemical CO2reduction reaction (CO2RR) to CO. Further improving their intrinsic activity and selectivity by tuning their N−M bond structures and coordination is limited. Herein, we expand the coordination environments of M−N−C catalysts by designing dual‐metal active sites. The Ni‐Fe catalyst exhibited the most efficient CO2RR activity and promising stability compared to other combinations. Advanced structural characterization and theoretical prediction suggest that the most active N‐coordinated dual‐metal site configurations are 2N‐bridged (Fe‐Ni)N6, in which FeN4and NiN4moieties are shared with two N atoms. Two metals (i.e., Fe and Ni) in the dual‐metal site likely generate a synergy to enable more optimal *COOH adsorption and *CO desorption than single‐metal sites (FeN4or NiN4) with improved intrinsic catalytic activity and selectivity. 
    more » « less
  2. ; ; ; ; ; ; ; ; (, Nature Communications)
    Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. 
    more » « less
  3. ; ; ; ; ; (, Angewandte Chemie International Edition)
    Abstract The complex structure of the catalytic active phase, and surface‐gas reaction networks have hindered understanding of the oxidative coupling of methane (OCM) reaction mechanism by supported Na2WO4/SiO2catalysts. The present study demonstrates, with the aid of in situ Raman spectroscopy and chemical probe (H2‐TPR, TAP and steady‐state kinetics) experiments, that the long speculated crystalline Na2WO4active phase is unstable and melts under OCM reaction conditions, partially transforming to thermally stable surface Na‐WOxsites. Kinetic analysis via temporal analysis of products (TAP) and steady‐state OCM reaction studies demonstrate that (i) surface Na‐WOxsites are responsible for selectively activating CH4to C2Hxand over‐oxidizing CHyto CO and (ii) molten Na2WO4phase is mainly responsible for over‐oxidation of CH4to CO2and also assists in oxidative dehydrogenation of C2H6to C2H4. These new insights reveal the nature of catalytic active sites and resolve the OCM reaction mechanism over supported Na2WO4/SiO2catalysts. 
    more » « less
  4. ; ; ; ; ; ; ; (, Proceedings of the National Academy of Sciences)
    While considerable knowledge exists about the enzymes pivotal for C4photosynthesis, much less is known about thecis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4enzymes for five different grass species. This study spans four C4species, covering three distinct photosynthetic subtypes:Zea maysandSorghum bicolor(NADP-dependent malic enzyme),Panicum miliaceum(NAD-dependent malic enzyme),Urochloa fusca(phosphoenolpyruvate carboxykinase), along with the C3outgroupOryza sativa. We studied thecis-regulatory landscape of enzymes essential across all C4species and those unique to C4subtypes, measuring cell-type-specific biases for C4enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4evolution. Besides promoter proximal ACRs, we found that, on average, C4genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution ofcis-regulation at these C4loci. This study illuminates the dynamic and complex nature ofcis-regulatory elements evolution in C4photosynthesis, particularly highlighting the intricatecis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3crop performance under changing climatic conditions. 
    more » « less
  5. ; ; ; ; ; ; (, Proceedings of the National Academy of Sciences)
    How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data forPseudomonas putidaketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email