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1.
Proc Natl Acad Sci U S A ; 112(12): 3704-9, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25775555

ABSTRACT

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.


Subject(s)
Carbon/chemistry , Protein Engineering/methods , Proteins/chemistry , Biomass , Biosynthetic Pathways , Carbon Cycle , Catalysis , Cloning, Molecular , Escherichia coli/enzymology , Formaldehyde/chemistry , Formates/chemistry , Magnetic Resonance Spectroscopy , Polymerase Chain Reaction , Software , Thermodynamics
2.
ACS Chem Biol ; 8(4): 749-57, 2013 Apr 19.
Article in English | MEDLINE | ID: mdl-23330600

ABSTRACT

The Morita-Baylis-Hillman reaction forms a carbon-carbon 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 ß-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.


Subject(s)
Proteins/metabolism , Catalysis , Kinetics , Molecular Dynamics Simulation , Proteins/chemistry , X-Ray Diffraction
3.
J Am Chem Soc ; 134(39): 16197-206, 2012 Oct 03.
Article in English | MEDLINE | ID: mdl-22871159

ABSTRACT

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 (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-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.


Subject(s)
Biocatalysis , Drug Design , Esterases/chemistry , Esterases/metabolism , Models, Molecular , Catalytic Domain , Esters , Hydrogen Bonding , Hydrolysis , Kinetics
4.
Nat Chem Biol ; 8(3): 294-300, 2012 Feb 05.
Article in English | MEDLINE | ID: mdl-22306579

ABSTRACT

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(-1) s(-1) 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.


Subject(s)
Adenosine Deaminase/metabolism , Computer Simulation , Computer-Aided Design , Metalloproteins/metabolism , Organophosphorus Compounds/metabolism , Zinc/chemistry , Adenosine Deaminase/chemistry , Animals , Biocatalysis , Catalytic Domain , Computational Biology , Hydrolysis , Metalloproteins/chemistry , Mice , Models, Molecular , Molecular Conformation , Organophosphorus Compounds/chemistry , Zinc/metabolism
5.
Science ; 329(5989): 309-13, 2010 Jul 16.
Article in English | MEDLINE | ID: mdl-20647463

ABSTRACT

The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.


Subject(s)
Carbon/chemistry , Computer-Aided Design , Enzymes/chemistry , Protein Engineering , Proteins/chemistry , Acrylamides/chemistry , Algorithms , Butadienes/chemistry , Catalysis , Catalytic Domain , Chemical Phenomena , Computer Simulation , Crystallography, X-Ray , Enzymes/genetics , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Mutagenesis , Protein Conformation , Proteins/genetics , Software , Stereoisomerism , Substrate Specificity
6.
Proc Natl Acad Sci U S A ; 106(23): 9215-20, 2009 Jun 09.
Article in English | MEDLINE | ID: mdl-19470646

ABSTRACT

Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme-substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein-ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-A Calpha RMSD of the computational model.


Subject(s)
Guanine Deaminase/chemistry , Protein Engineering/methods , Algorithms , Crystallography, X-Ray , Guanine Deaminase/metabolism , Humans , Models, Molecular , Substrate Specificity , Triazines/metabolism
7.
Gastroenterology ; 135(2): 410-8, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18519043

ABSTRACT

BACKGROUND & AIMS: Telomere shortening is implicated in cancer and aging and might link these 2 biologic events. We explored this hypothesis in ulcerative colitis (UC), a chronic inflammatory disease that predisposes to colorectal cancer and in which shorter telomeres have been associated with chromosomal instability and tumor progression. METHODS: Telomere length was measured by quantitative polymerase chain reaction in colonocytes and leukocytes of 2 different sets of UC patients and compared with normal controls across a wide range of ages. For a subset of patients, telomere length was measured in epithelium and stroma of right and left colon biopsy specimens. A third set of biopsy specimens was analyzed for phosphorylation of histone H2AX (gammaH2AX), a DNA damage signal, by immunofluorescence and for telomere length by quantitative fluorescence in situ hybridization. Relationships between telomere length, gammaH2AX intensity, age, disease duration, and age of disease onset were explored. RESULTS: Colonocyte telomeres shorten with age almost twice as rapidly in UC patients as in normal controls. This extensive shortening occurs within approximately 8 years of disease duration. Leukocyte telomeres are slightly shorter in UC patients than in controls, but telomeres of colon stromal cells are unaffected. gammaH2AX intensity is higher in colonocytes of UC patients than in controls and is not dependent on age or telomere length. CONCLUSIONS: Colonocytes of UC patients show premature shortening of telomeres, which might explain the increased and earlier risk of cancer in this disease. Shorter leukocyte telomeres and increased gammaH2AX in colonocytes might reflect oxidative damage secondary to inflammation.


Subject(s)
Aging/genetics , Colitis, Ulcerative/genetics , Colon/metabolism , Colorectal Neoplasms/genetics , DNA Damage , Telomere/metabolism , Adolescent , Adult , Age Distribution , Age Factors , Aged , Aging/metabolism , Aging/pathology , Child , Child, Preschool , Colitis, Ulcerative/complications , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/pathology , Colon/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cross-Sectional Studies , Female , Fluorescent Antibody Technique , Histones/metabolism , Humans , In Situ Hybridization, Fluorescence , Intestinal Mucosa/metabolism , Leukocytes/metabolism , Male , Middle Aged , Phosphorylation , Polymerase Chain Reaction , Stromal Cells/metabolism , Up-Regulation
8.
Nature ; 453(7192): 190-5, 2008 May 08.
Article in English | MEDLINE | ID: mdl-18354394

ABSTRACT

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.


Subject(s)
Computer Simulation , Directed Molecular Evolution/methods , Enzymes/chemistry , Enzymes/metabolism , Protein Engineering/methods , Algorithms , Amino Acid Motifs , Binding Sites/genetics , Catalysis , Computational Biology , Crystallography, X-Ray , Drug Design , Drug Evaluation, Preclinical , Enzymes/genetics , Kinetics , Models, Chemical , Models, Molecular , Quantum Theory , Sensitivity and Specificity
9.
Science ; 319(5868): 1387-91, 2008 Mar 07.
Article in English | MEDLINE | ID: mdl-18323453

ABSTRACT

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.


Subject(s)
Aldehyde-Lyases/chemistry , Algorithms , Aldehyde-Lyases/metabolism , Binding Sites , Catalysis , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Protein Conformation , Protein Engineering
10.
Cancer Epidemiol Biomarkers Prev ; 16(12): 2649-55, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18086770

ABSTRACT

PURPOSE: Leukocyte telomere length has gained attention as a marker of oxidative damage and age-related diseases, including cancer. We hypothesize that leukocyte telomere length might be able to predict future risk of cancer and examined this in a cohort of patients with Barrett's esophagus, who are at increased risk of esophageal adenocarcinoma and thus were enrolled in a long-term cancer surveillance program. PATIENTS AND METHODS: In this prospective study, telomere length was measured by quantitative PCR in baseline blood samples in a cohort of 300 patients with Barrett's esophagus followed for a mean of 5.8 years. Leukocyte telomere length hazard ratios (HR) for risk of esophageal adenocarcinoma were calculated using multivariate Cox models. RESULTS: Shorter telomeres were associated with increased esophageal adenocarcinoma risk (age-adjusted HR between top and bottom quartiles of telomere length, 3.45; 95% confidence interval, 1.35-8.78; P = 0.009). This association was still significant when individually or simultaneously adjusted for age, gender, nonsteroidal anti-inflammatory drug (NSAID) use, cigarette smoking, and waist-to-hip ratio (HR, 4.18; 95% confidence interval, 1.60-10.94; P = 0.004). The relationship between telomere length and cancer risk was particularly strong among NSAID nonusers, ever smokers, and patients with low waist-to-hip ratio. CONCLUSION: Leukocyte telomere length predicts risk of esophageal adenocarcinoma in patients with Barrett's esophagus independently of smoking, obesity, and NSAID use. These results show the ability of leukocyte telomere length to predict the risk of future cancer and suggest that it might also have predictive value in other cancers arising in a setting of chronic inflammation.


Subject(s)
Adenocarcinoma/pathology , Barrett Esophagus/pathology , Biomarkers, Tumor/analysis , Esophageal Neoplasms/pathology , Leukocytes/physiology , Telomere/metabolism , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Humans , Polymerase Chain Reaction , Precancerous Conditions/pathology , Risk Factors , Smoking/adverse effects , Waist-Hip Ratio
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