Marine cyanobacteria are infected by phages whose genomes encode ferredoxin (Fd) electron carriers. These Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases ...that enhance viral fitness, but it is unclear how the biophysical properties and partner specificities of phage Fds relate to those of photosynthetic organisms. Here, results of a bioinformatics analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects the cyanobacterium Prochlorococcus marinus, revealed high levels of similarity to cyanobacterial Fds (root mean square deviations of ≤0.5 Å). Additionally, pssm2-Fd exhibited a low midpoint reduction potential (–336 mV versus a standard hydrogen electrode), similar to other photosynthetic Fds, although it had lower thermostability (Tm = 28 °C) than did many other Fds. When expressed in an Escherichia coli strain deficient in sulfite assimilation, pssm2-Fd complemented bacterial growth when coexpressed with a P. marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high levels of structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterially encoded oxidoreductases.
Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more ...difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple “movies” to be taken of proteins in action, and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life’s machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.
Highlights ► Glycosyltransferases (GTs) are an important class of enzyme as both drug targets and synthetic catalysts. ► Two predominant GT folds (GT-A and GT-B) enable the connection between a vast ...range of sugar donors and acceptors representing a wide array of regiospecificities and stereospecificities. ► Although few ‘general’ rules exist, rational engineering has led to GTs with altered specificities (sugar donor and/or acceptors). ► In the context of engineered GTs, an enhancement of promiscuity often correlates with an increase in overall proficiency.
Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene ...duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to α-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.
The phytochrome superfamily encompasses a diverse collection of photochromic photoreceptors in plants and microorganisms that employ a covalently linked bilin cradled in a ...cGMP-phosphodiesterase/adenylyl-cyclase/FhlA (GAF) domain to detect light. Whereas most interconvert between red- and far-red-light-absorbing states, cyanobacteria also express variants called cyanobacteriochromes (CBCRs) that modify bilin absorption to collectively perceive the entire visible spectrum. Here, we present two X-ray crystallographic structures of the GAF domain from the blue/green photochromic CBCR PixJ from Thermosynechococcus elongatus. These structures confirm the hypothesis that CBCRs variably manipulate the chromophore π-conjugation system through isomerization and a second thioether linkage, in this case involving the bilin C10 carbon and Cys494 within a DXCF sequence characteristic of blue/green CBCRs. Biochemical studies support a mechanism for photoconversion whereby the second linkage ruptures on route to the green-light-absorbing state. Collectively, the TePixJ(GAF) models illustrate the remarkable structural and photochemical versatility among phytochromes and CBCRs in driving light perception.
▸ The Te-PixJ structure reveals the positions of the two DXCF-CBCR thioether linkages ▸ For blue/green photocycling, Te-PixJ utilizes a phycoviolobilin chromophore ▸ Rupture of the noncanonical DXCF thioether coincides with Pb/Pg photoconversion ▸ Te-PixJ bilin-binding and photochemistry diverge greatly from canonical phytochromes
Phytochromes use a thioether-linked bilin to interconvert between red- and far-red light-absorbing states. Burgie et al. describe structures of PixJ, a cyanobacteriochrome, and how it enforces a blue/green photocycles by manipulating bilin π-conjugation through isomerization and a photo-labile thioether-linkage.
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary ...techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.
Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, ...whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.
Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of ...cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph Iβ to IIII, which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose IIII, which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ∼50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60–70% lower maximum surface-bound cellulase capacity. The enhancement in apparent cellulase activity was attributed to the “amorphous-like” nature of the cellulose IIII fibril surface that facilitated easier glucan chain extraction. Unrestricted substrate accessibility to active-site clefts of certain endocellulase families further accelerated deconstruction of cellulose IIII. Structural and dynamical features of cellulose IIII, revealed by MD simulations, gave additional insights into the role of cellulose crystal structure on fibril surface hydration that influences interfacial enzyme binding. Subtle alterations within the cellulose hydrogen bond network provide an attractive way to enhance its deconstruction and offer unique insight into the nature of cellulose recalcitrance. This approach can lead to unconventional pathways for development of novel pretreatments and engineered cellulases for cost-effective biofuels production.
This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to ...a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug.
We report picosecond time-resolved x-ray diffraction from the myoglobin (Mb) mutant in which$Leu^{29}$is replaced by Phe (L29F mutant). The frame-by-frame structural evolution, resolved to 1.8 ...angstroms, allows one to literally "watch" the protein as it executes its function. Time-resolved mid-infrared spectroscopy of flash-photolyzed L29F MbCO revealed a short-lived CO intermediate whose 140-ps lifetime is shorter than that found in wild-type protein by a factor of 1000. The electron density maps of the protein unveil transient conformational changes far more dramatic than the structural differences between the carboxy and deoxy states and depict the correlated side-chain motion responsible for rapidly sweeping CO away from its primary docking site.
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