Search NASA⌕ Search

SEARCH · Search NASA

Results for “enzyme evolution”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

291 records · Page 17

Photoenzymatic Csp 3 –Csp 3 bond formation via enzyme-templated radical–radical coupling

Cross-couplings are essential reactions in modern chemical synthesis, enabling the rapid construction of complex molecules from simple precursors. Transition metal catalysts are prized for these transformations because their reactivity and selectivity can be tuned via judicious selection of the metal and ligand. Although enzymes offer analogous opportunities for tuning via protein engineering, their application to cross-coupling remains limited, as nature relies on alternative paradigms for building molecular complexity. Here, we report the cross-coupling of alkyl halides and benzylic carboxylic acids using an engineered flavin-dependent lactate monooxygenase—a photoenzyme. The enzyme achieves this feat by exploiting the redox versatility of the flavin cofactor. Stoichiometric experiments, ultrafast spectroscopy, and computational studies support a mechanism in which photoexcited flavin quinone initiates the reaction via oxidative decarboxylation to generate a benzylic radical. The resulting flavin semiquinone can reduce the alkyl halide to form a second organic radical within the protein active site, which rapidly engages in C(sp 3 )–C(sp 3 ) bond formation. A variant was engineered to control the stereochemical outcome of this radical–radical coupling event, highlighting the ability of the protein to alter the energetic barrier for a mechanistic step that is traditionally understood to be near barrierless. This work demonstrates that the scope for nonnative reaction mechanisms in biocatalysis far exceeds previously established bounds and has potential to solve a variety of reactivity challenges in cross-coupling chemistry.

biocatalysis↗

MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.

Novosphingobium↗

Estimating the Timing of Early Simian-Human Immunodeficiency Virus Infections: a Comparison between Poisson Fitter and BEAST

Many HIV prevention strategies are currently under consideration where it is highly informative to know the study participants’ times of infection. These can be estimated using viral sequence data sampled early in infection. However, there are several scenarios that, if not addressed, can skew timing estimates. These include multiple transmitted/founder (TF) viruses, APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like)-mediated mutational enrichment, and recombination. Here, we suggest a pipeline to identify these problems and resolve the biases that they introduce. We then compare two modeling strategies to obtain timing estimates from sequence data. The first, Poisson Fitter (PF), is based on a Poisson model of random accumulation of mutations relative to the TF virus (or viruses) that established the infection. The second uses a coalescence-based phylogenetic strategy as implemented in BEAST. The comparison is based on timing predictions using plasma viral RNA (cDNA) sequence data from 28 simian-human immunodeficiency virus (SHIV)-infected animals for which the exact day of infection is known. In this particular setting, based on nucleotide sequences from samples obtained in early infection, the Poisson method yielded more accurate, more precise, and unbiased estimates for the time of infection than did the explored implementations of BEAST. The inference of the time of infection is a critical parameter in testing the efficacy of clinical interventions in protecting against HIV-1 infection. For example, in clinical trials evaluating the efficacy of passively delivered antibodies (Abs) for preventing infections, accurate time of infection data are essential for discerning levels of the Abs required to confer protection, given the natural Ab decay rate in the human body. In such trials, genetic sequences from early in the infection are regularly sampled from study participants, generally prior to immune selection, when the viral population is still expanding and genetic diversity is low. In this particular setting of early viral growth, the Poisson method is superior to the alternative approach based on coalescent methods. This approach can also be applied in human vaccine trials, where accurate estimates of infection times help ascertain if vaccine-elicited immune protection wanes over time.

59 BASIC BIOLOGICAL SCIENCES↗