Elucidation of the folding pathway of a circular permutant of topologically knotted YbeA by tryptophan substitutions.

CP74 is an engineered circular permutant of a deep trefoil knotted SpoU-TrmD (SPOUT) RNA methyl transferase protein YbeA from E. coli. We have previously established that the circular permutation unties the knotted topology of YbeA and CP74 forms a domain-swapped dimer with a large dimeric interface of ca. 4600 Å2. To understand the impact of domain-swapping and the newly formed hinge region joining the two folded domains on the folding and stability of CP74, the five equally spaced tryptophan residues were individually substituted into phenylalanine to monitor their conformational and stability changes by a battery of biophysical tools. Far-UV circular dichroism, intrinsic fluorescence, and small-angle X-ray scattering dictated minimal global conformational perturbations to the native structures in the tryptophan variants. The structures of the tryptophan variants also showed the conservation of the domain-swapped ternary structure with the exception that the W72F exhibited significant asymmetry in the α-helix 5. Comparative global thermal and chemical stability analyses indicated the pivotal role of W100 in the folding of CP74 followed by W19 and W72. Solution-state NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry further revealed the accumulation of a native-like intermediate state in which the hinge region made important contributions to maintain the domain-swapped ternary structure of CP74.

An essential role of acetyl coenzyme A in the catalytic cycle of insect arylalkylamine N-acetyltransferase.

Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine N-acetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Here, two ternary complexes of dopamine N-acetyltransferase (Dat) before and after N-acetylation were solved at 1.28 Å and 1.36 Å resolution, respectively. Combined with the structures of Dat in apo form and Ac-CoA bound form, we addressed each stage in the catalytic cycle. Isothermal titration calorimetry (ITC), crystallography, and nuclear magnetic resonance spectroscopy (NMR) were utilized to analyze the product release. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA.

Untying a Knotted SPOUT RNA Methyltransferase by Circular Permutation Results in a Domain-Swapped Dimer.

YbeA from E. coli is a trefoil-knotted SpoU-TrmD (SPOUT) RNA methyltransferase. While its knotted motif plays a key functional role, it is unclear how the knotted topology emerged from evolution. Here, we reverse-engineered an unknotted circular permutant (CP) of YbeA by introducing a new opening at the knotting loop. The resulting CP folded into an unexpected domain-swapped dimer. Untying the knotted loop abrogated its function, perturbed its folding stability and kinetics, and induced allosteric dynamic changes. We speculated that the knotted loop of YbeA is under tension to keep the cofactor in a high-energy configuration while keeping the threading C-terminal helix being knotted. Circular permutation released the mechanical strain thereby allowing the spring-loaded threading helix to flip, to relax, and to form a domain-swapped dimer. Being knotted may be the consequence of selection pressure for the unique structure-function relationship of the SPOUT superfamily that exists in all kingdoms of life.

Untying a Protein Knot by Circular Permutation.

Topologically knotted proteins are tantalizing examples of how polypeptide chains can explore complex free energy landscapes to efficiently attain defined knotted conformations. The evolution trails of protein knots, however, remain elusive. We used circular permutation to change an evolutionally conserved topologically knotted SPOUT RNA methyltransferase into an unknotted form. The unknotted variant adopted the same three-dimensional structure and oligomeric state as its knotted parent, but its folding stability was markedly reduced with accelerated folding kinetics and its ligand binding was abrogated. Our findings support the hypothesis that the universally conserved knotted topology of the SPOUT superfamily evolved from unknotted forms through circular permutation under selection pressure for folding robustness and, more importantly, for functional requirements associated with the knotted structural element.

Well-tolerated Spirulina extract inhibits influenza virus replication and reduces virus-induced mortality.

Influenza is one of the most common human respiratory diseases, and represents a serious public health concern. However, the high mutability of influenza viruses has hampered vaccine development, and resistant strains to existing anti-viral drugs have also emerged. Novel anti-influenza therapies are urgently needed, and in this study, we describe the anti-viral properties of a Spirulina (Arthrospira platensis) cold water extract. Anti-viral effects have previously been reported for extracts and specific substances derived from Spirulina, and here we show that this Spirulina cold water extract has low cellular toxicity, and is well-tolerated in animal models at one dose as high as 5,000 mg/kg, or 3,000 mg/kg/day for 14 successive days. Anti-flu efficacy studies revealed that the Spirulina extract inhibited viral plaque formation in a broad range of influenza viruses, including oseltamivir-resistant strains. Spirulina extract was found to act at an early stage of infection to reduce virus yields in cells and improve survival in influenza-infected mice, with inhibition of influenza hemagglutination identified as one of the mechanisms involved. Together, these results suggest that the cold water extract of Spirulina might serve as a safe and effective therapeutic agent to manage influenza outbreaks, and further clinical investigation may be warranted.

Microalgae-based biorefinery--from biofuels to natural products.

The potential for biodiesel production from microalgal lipids and for CO2 mitigation due to photoautotrophic growth of microalgae have recently been recognized. Microalgae biomass also has other valuable components, including carbohydrates, long chain fatty acids, pigments and proteins. The microalgae-based carbohydrates consist mainly of cellulose and starch without lignin; thus they can be ready carbon source for the fermentation industry. Some microalgae can produce long chain fatty acids (such as DHA and EPA) as valuable health food supplements. In addition, microalgal pigments and proteins have considerable potential for many medical applications. This review article presents comprehensive information on the current state of these commercial applications, as well as the utilization and characteristics of the microalgal components, in addition to the key factors and challenges that should be addressed during the production of these materials, and thus provides a useful report that can aid the development of an efficient microalgae-based biorefinery process.

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalytic bilin attachment.

Allophycocyanin (APC) is one of the phycobiliproteins expressed in cyanobacteria. Phycobiliproteins contain a covalently bound chromophore, and thus, they are valuable as fluorescent probes. Biosynthesis of a functional phycobiliprotein is achieved by a bilin attachment process between the chromophore and apoprotein. Chromophore lyases are necessary to catalyze the chromophorylation of cyanobacterial phycobiliproteins, such as C-phycocyanin, and phycoerythrocyanin. To identify the lyase that catalyzes the chromophorylation of the APC alpha-subunit (ApcA), we searched the entire genomes of two cyanobacteria, Synechocystis sp. PCC6803 and Anabaena sp. PCC 7120; however, these genomes do not appear to encode an APC-specific chromophore lyase. In this study, chromophorylated ApcA (chromo-ApcA) was obtained via a spontaneous bilin attachment reaction. The absorption and fluorescence characteristics of chromo-ApcA were similar to those of the native APC alpha-subunit. The extent of chromophore attachment to apo-ApcA was comparable to that of the lyase-catalyzed reactions for other phycobiliproteins. These results indicate that ApcA has autocatalytic bilin:biliprotein lyase activity.