1,684 research outputs found
A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases
The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal–Structural Analysis and Library Design (CSR-SALAD)
Spin-Orbit and Tensor Forces in Heavy-quark Light-quark Mesons: Implications of the New Ds state at 2.32 GeV
We consider the spectroscopy of heavy-quark light-quark mesons with a simple
model based on the non-relativistic reduction of vector and scalar exchange
between fermions. Four forces are induced: the spin-orbit forces on the light
and heavy quark spins, the tensor force, and a spin-spin force. If the vector
force is Coulombic, the spin-spin force is a contact interaction, and the
tensor force and spin-orbit force on the heavy quark to order are
directly proportional. As a result, just two independent parameters
characterize these perturbations. The measurement of the masses of three p-wave
states suffices to predict the mass of the fourth. This technique is applied to
the system, where the newly discovered state at 2.32 GeV provides the
third measured level, and to the system. The mixing of the two
p-wave states is reflected in their widths and provides additional constraints.
The resulting picture is at odds with previous expectations and raises new
puzzles.Comment: 6 pages, 1 figur
4S-Hydroxylation of insulin at ProB28 accelerates hexamer dissociation and delays fibrillation
Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of non-canonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins
Artificial domain duplication replicates evolutionary history of ketol-acid reductoisomerases
The duplication of protein structural domains has been proposed as a common mechanism for the generation of new protein folds. A particularly interesting case is the class II ketol-acid reductoisomerase (KARI), which putatively arose from an ancestral class I KARI by duplication of the C-terminal domain and corresponding loss of obligate dimerization. As a result, the class II enzymes acquired a deeply embedded figure-of-eight knot. To test this evolutionary hypothesis we constructed a novel class II KARI by duplicating the C-terminal domain of a hyperthermostable class I KARI. The new protein is monomeric, as confirmed by gel filtration and x-ray crystallography, and has the deeply-knotted class II KARI fold. Surprisingly, its catalytic activity is nearly unchanged from the parent KARI. This provides strong evidence in support of domain duplication as the mechanism for the evolution of the class II KARI fold and demonstrates the ability of domain duplication to generate topological novelty in a function-neutral manner
General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH
To date, efforts to switch the cofactor specificity of oxidoreductases
from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide
adenine dinucleotide (NADH) have been made on a case-by-
case basis with varying degrees of success. Here we present a
straightforward recipe for altering the cofactor specificity of a class of
NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases
(KARIs). Combining previous results for an engineered NADH-dependent
variant of Escherichia coli KARI with available KARI crystal
structures and a comprehensive KARI-sequence alignment, we identified
key cofactor specificity determinants and used this information to
construct five KARIs with reversed cofactor preference. Additional directed
evolution generated two enzymes having NADH-dependent
catalytic efficiencies that are greater than the wild-type enzymes with
NADPH. High-resolution structures of a wild-type/variant pair reveal
the molecular basis of the cofactor switch
Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases
Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site
Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models
The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate
Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore
By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems
Structural, Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE
A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild-type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB′1 helix of the protein to seal the binding pocket
Two-photon final states in peripheral heavy ion collisions
We discuss processes leading to two photon final states in peripheral heavy
ion collisions at RHIC. Due to the large photon luminosity we show that the
continuum subprocess can be observed with a
large number of events. We study this reaction when it is intermediated by a
resonance made of quarks or gluons and discuss its interplay with the continuum
process, verifying that in several cases the resonant process ovewhelms the
continuum one. It is also investigated the possibility of observing a scalar
resonance (the meson) in this process. Assuming for the the
mass and total decay width values recently reported by the E791 Collaboration
we show that RHIC may detect this particle in its two photon decay mode if its
partial photonic decay width is of the order of the ones discussed in the
literature.Comment: 10 pages, 8 figure
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