Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase.

UNLABELLED: Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. IMPORTANCE: STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into the virion during infection. The experiments described here highlight the elements of this enzyme that are essential for proper function and also provide supporting evidence that B204 is present in the mature STIV virion.

Phylogenetic and kinetic characterization of a suite of dehydrogenases from a newly isolated bacterium, strain SG61-1L, that catalyze the turnover of guaiacylglycerol-ß-guaiacyl ether stereoisomers.

Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-ß-guaiacyl ether (GGE), which contains a key ß-O-4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities.

Adapting protein sequences for optimized therapeutic efficacy.

Therapeutic proteins alleviate disease pathology by supplementing missing or defective native proteins, sequestering superfluous proteins, or by acting through designed non-natural mechanisms. Although therapeutic proteins often have the same amino acid sequence as their native counterpart, their maturation paths from expression to the site of physiological activity are inherently different, and optimizing protein sequences for properties that 100s of millions of years of evolution did not need to address presents an opportunity to develop better biological treatments. Because therapeutic proteins are inherently non-natural entities, optimization for their desired function should be considered analogous to that of small molecule drug candidates, which are optimized through expansive combinatorial variation by the medicinal chemist. Here, we review recent successes and challenges of protein engineering for optimized therapeutic efficacy.

Bisabolyl-derived sesquiterpenes from tobacco 5-epi-aristolochene synthase-catalyzed cyclization of (2Z,6E)-farnesyl diphosphate.

We report the structures and stereochemistry of seven bisabolyl-derived sesquiterpenes arising from an unprecedented 1,6-cyclization (cisoid pathway) efficiently catalyzed by tobacco 5-epi-aristolochene synthase (TEAS). The use of (2Z,6E)-farnesyl diphosphate as an alternate substrate for recombinant TEAS resulted in a robust enzymatic cyclization to an array of products derived exclusively (>/=99.5%) from the cisoid pathway, whereas these same products account for ca. 2.5% of the total hydrocarbons obtained using (2E,6E)-farnesyl diphosphate. Chromatographic fractionations of extracts from preparative incubations with the 2Z,6E substrate afforded, in addition to the acyclic allylic alcohols (2Z,6E)-farnesol (6.7%) and nerolidol (3.6%), five cyclic sesquiterpene hydrocarbons and two cyclic sesquiterpene alcohols: (+)-2-epi-prezizaene (44%), (-)-alpha-cedrene (21.5%), (R)-(-)-beta-curcumene (15.5%), alpha-acoradiene (3.9%), 4-epi-alpha-acoradiene (1.3%), and equal amounts of alpha-bisabolol (1.8%) and epi-alpha-bisalolol (1.8%). The structures, stereochemistry, and enantiopurities were established by comprehensive spectroscopic analyses, optical rotations, chemical correlations with known sesquiterpenes, comparisons with literature data, and GC analyses. The major product, (+)-2-epi-prezizaene, is structurally related to the naturally occurring tricyclic alcohol, jinkohol (2-epi-prezizaan-7beta-ol). Cisoid cyclization pathways are proposed by which all five sesquiterpene hydrocarbons are derived from a common (7R)-beta-bisabolyl(+)/pyrophosphate(-) ion pair intermediate. The implications of the "cisoid" catalytic activity of TEAS are discussed.

Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases.

Throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. Catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. Here we report the first systematic quantitative characterization of the catalytic landscape underlying the evolution of sesquiterpene chemical diversity. On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus, we created a library of all possible residue combinations (2(9) = 512) in the N. tabacum enzyme. The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities. Quantitative comparisons indicated context dependence for mutational effects--epistasis--in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

European Immunogenicity Platform 11th Open Scientific Symposium on immunogenicity of biopharmaceuticals.

Given the expanding number of complex therapeutic protein drugs and advanced therapy medicinal products that are being developed, improving our ability to assess the potential immunogenicity of biologics is critical to ensuring treatment efficacy and patient safety. In this context, the European Immunogenicity Platform annual meeting provides opportunities for experts from industry and academia, regulators and clinicians to convene and discuss immunogenicity assessment methods and tools. This report summarizes the key messages on immunogenicity testing, prediction, clinical relevance and advanced therapy medicinal products discussed at the 11th Open Scientific European Immunogenicity Platform Symposium on Immunogenicity of Biopharmaceuticals, Lisbon, Portugal, 17-19 February 2020.

Archaeal Viruses: Diversity, Replication, and Structure.

The Archaea-and their viruses-remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was described over 40 years ago, only 117 archaeal viruses have been discovered to date. Despite this small number, these viruses have painted a portrait of enormous morphological and genetic diversity. For example, research centered around the various steps of the archaeal virus life cycle has led to the discovery of unique mechanisms employed by archaeal viruses during replication, maturation, and virion release. In many instances, archaeal virus proteins display very low levels of sequence homology to other proteins listed in the public database, and therefore, structural characterization of these proteins has played an integral role in functional assignment. These structural studies have not only provided insights into structure-function relationships but have also identified links between viruses across all three domains of life.

A survey of protein structures from archaeal viruses.

Viruses that infect the third domain of life, Archaea, are a newly emerging field of interest. To date, all characterized archaeal viruses infect archaea that thrive in extreme conditions, such as halophilic, hyperthermophilic, and methanogenic environments. Viruses in general, especially those replicating in extreme environments, contain highly mosaic genomes with open reading frames (ORFs) whose sequences are often dissimilar to all other known ORFs. It has been estimated that approximately 85% of virally encoded ORFs do not match known sequences in the nucleic acid databases, and this percentage is even higher for archaeal viruses (typically 90%-100%). This statistic suggests that either virus genomes represent a larger segment of sequence space and/or that viruses encode genes of novel fold and/or function. Because the overall three-dimensional fold of a protein evolves more slowly than its sequence, efforts have been geared toward structural characterization of proteins encoded by archaeal viruses in order to gain insight into their potential functions. In this short review, we provide multiple examples where structural characterization of archaeal viral proteins has indeed provided significant functional and evolutionary insight.

Discovery of a metabolic alternative to the classical mevalonate pathway.

Eukarya, Archaea, and some Bacteria encode all or part of the essential mevalonate (MVA) metabolic pathway clinically modulated using statins. Curiously, two components of the MVA pathway are often absent from archaeal genomes. The search for these missing elements led to the discovery of isopentenyl phosphate kinase (IPK), one of two activities necessary to furnish the universal five-carbon isoprenoid building block, isopentenyl diphosphate (IPP). Unexpectedly, we now report functional IPKs also exist in Bacteria and Eukarya. Furthermore, amongst a subset of species within the bacterial phylum Chloroflexi, we identified a new enzyme catalyzing the missing decarboxylative step of the putative alternative MVA pathway. These results demonstrate, for the first time, a functioning alternative MVA pathway. Key to this pathway is the catalytic actions of a newly uncovered enzyme, mevalonate phosphate decarboxylase (MPD) and IPK. Together, these two discoveries suggest that unforeseen variation in isoprenoid metabolism may be widespread in nature. DOI: http://dx.doi.org/10.7554/eLife.00672.001.

Stereochemical basis for engineered pyrrolysyl-tRNA synthetase and the efficient in vivo incorporation of structurally divergent non-native amino acids.

Unnatural amino acids (Uaas) can be translationally incorporated into proteins in vivo using evolved tRNA/aminoacyl-tRNA synthetase (RS) pairs, affording chemistries inaccessible when restricted to the 20 natural amino acids. To date, most evolved RSs aminoacylate Uaas chemically similar to the native substrate of the wild-type RS; these conservative changes limit the scope of Uaa applications. Here, we adapt Methanosarcina mazei PylRS to charge a noticeably disparate Uaa, O-methyl-l-tyrosine (Ome). In addition, the 1.75 Å X-ray crystal structure of the evolved PylRS complexed with Ome and a non-hydrolyzable ATP analogue reveals the stereochemical determinants for substrate selection. Catalytically synergistic active site mutations remodel the substrate-binding cavity, providing a shortened but wider active site. In particular, mutation of Asn346, a residue critical for specific selection and turnover of the Pyl chemical core, accommodates different side chains while the central role of Asn346 in aminoacylation is rescued through compensatory hydrogen bonding provided by A302T. This multifaceted analysis provides a new starting point for engineering PylRS to aminoacylate a significantly more diverse selection of Uaas than previously anticipated.

Mutation of archaeal isopentenyl phosphate kinase highlights mechanism and guides phosphorylation of additional isoprenoid monophosphates.

The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg(2+)-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0-2.8 A. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nepsilon(2) nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors.

Structural elucidation of cisoid and transoid cyclization pathways of a sesquiterpene synthase using 2-fluorofarnesyl diphosphates.

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (cis,trans)-FPP (1b) to exclusively (>/=99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP (1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed.