12 research outputs found
Computational protein design enables a novel one-carbon assimilation pathway.
We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway
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Computational protein design enables a novel one-carbon assimilation pathway.
We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway
Computational protein design enables a novel one-carbon assimilation pathway
We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway
Computational Design of Enone-Binding Proteins with Catalytic Activity for the MoritaâBaylisâHillman Reaction
The MoritaâBaylisâHillman reaction forms
a carbonâcarbon
bond between the α-carbon of a conjugated carbonyl compound
and a carbon electrophile. The reaction mechanism involves Michael
addition of a nucleophile catalyst at the carbonyl ÎČ-carbon,
followed by bond formation with the electrophile and catalyst disassociation
to release the product. We used Rosetta to design 48 proteins containing
active sites predicted to carry out this mechanism, of which two show
catalytic activity by mass spectrometry (MS). Substrate labeling measured
by MS and site-directed mutagenesis experiments show that the designed
active-site residues are responsible for activity, although rate acceleration
over background is modest. To characterize the designed proteins,
we developed a fluorescence-based screen for intermediate formation
in cell lysates, carried out microsecond molecular dynamics simulations,
and solved X-ray crystal structures. These data indicate a partially
formed active site and suggest several clear avenues for designing
more active catalysts
Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis
Nucleophilic catalysis is a general strategy for accelerating
ester
and amide hydrolysis. In natural active sites, nucleophilic elements
such as catalytic dyads and triads are usually paired with oxyanion
holes for substrate activation, but it is difficult to parse out the
independent contributions of these elements or to understand how they
emerged in the course of evolution. Here we explore the minimal requirements
for esterase activity by computationally designing artificial catalysts
using catalytic dyads and oxyanion holes. We found much higher success
rates using designed oxyanion holes formed by backbone NH groups rather
than by side chains or bridging water molecules and obtained four
active designs in different scaffolds by combining this motif with
a Cys-His dyad. Following active site optimization, the most active
of the variants exhibited a catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub>) of 400 M<sup>â1</sup> s<sup>â1</sup> for the cleavage of a <i>p</i>-nitrophenyl
ester. Kinetic experiments indicate that the active site cysteines
are rapidly acylated as programmed by design, but the subsequent slow
hydrolysis of the acyl-enzyme intermediate limits overall catalytic
efficiency. Moreover, the Cys-His dyads are not properly formed in
crystal structures of the designed enzymes. These results highlight
the challenges that computational design must overcome to achieve
high levels of activity
Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k cat/K m) of ~10 4 M â1 s â1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R P isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities. The redeployment of catalytic machinery in naturally occurring enzyme active sites for noncognate reactions holds considerable promise as a method for obtaining new biocatalysts 1,2. Alterations of substrate specificity and stereospecificity and the enhancement of preexisting promiscuous activities of enzymes have been accomplished by library-based directed evolution approaches 3. However, introducing completely new catalytic activities remains