Genetics re-establish the utility of 2-methylhopanes as cyanobacterial biomarkers before 750 million years ago.

Fossilized lipids offer a rare glimpse into ancient ecosystems. 2-Methylhopanes in sedimentary rocks were once used to infer the importance of cyanobacteria as primary producers throughout geological history. However, the discovery of hopanoid C-2 methyltransferase (HpnP) in Alphaproteobacteria led to the downfall of this molecular proxy. In the present study, we re-examined the distribution of HpnP in a new phylogenetic framework including recently proposed candidate phyla and re-interpreted a revised geological record of 2-methylhopanes based on contamination-free samples. We show that HpnP was probably present in the last common ancestor of cyanobacteria, while the gene appeared in Alphaproteobacteria only around 750 million years ago (Ma). A subsequent rise of sedimentary 2-methylhopanes around 600 Ma probably reflects the expansion of Alphaproteobacteria that coincided with the rise of eukaryotic algae-possibly connected by algal dependency on microbially produced vitamin B12. Our findings re-establish 2-methylhopanes as cyanobacterial biomarkers before 750 Ma and thus as a potential tool to measure the importance of oxygenic cyanobacteria as primary producers on early Earth. Our study illustrates how genetics can improve the diagnostic value of biomarkers and refine the reconstruction of early ecosystems.

Protection of Catalytic Cofactors by Polypeptides as a Driver for the Emergence of Primordial Enzymes.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Oral Delivery of Nanoparticles Carrying Ancestral Uricase Enzyme Protects against Hyperuricemia in Knockout Mice.

The therapeutic value of delivering recombinant uricase to human patients has been appreciated for decades. The development of therapeutic uricases has been hampered by the fact that humans do not encode an endogenous uricase and therefore most recombinant forms of the protein are recognized as foreign by the immune system and are therefore highly immunogenic. In order to both shield and stabilize the active enzyme, we encapsulated a functional ancestral uricase in recombinant, noninfectious Qß capsid nanoparticles and characterized its catalytic activity. Oral delivery of the nanoparticles moderated key symptoms of kidney dysfunction in uricase-knockout mice by lowering uric acid levels. Histological kidney samples of the treated mice suggest that delivery of recombinant uricase had a protective effect against the destructive effects of uric acid that lead to renal failure caused by hyperuricemia.

Protection of catalytic cofactors by polypeptides as a driver for the emergence of primordial enzymes.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Do thrifty genes exist? Revisiting uricase.

Sixty years ago, the geneticist James Neel proposed that the epidemics of obesity and diabetes today may have evolutionary roots. Specifically, he suggested that our ancestors may have accumulated mutations during periods of famine that provided a survival advantage at that time. However, the presence of this "thrifty genotype" in today's world, where food is plentiful, would predispose us to obesity and diabetes. The "thrifty gene" hypothesis, attractive to some, has been challenged over the years. The authors have previously postulated that the loss of the uricase gene, resulting in a rise in serum and intracellular uric acid levels, satisfies the criteria of a thrifty genotype mutation. This paper reviews and brings up-to-date the evidence supporting the hypothesis and discusses the current arguments that challenge this hypothesis. Although further studies are needed to test the hypothesis, the evidence supporting a loss of uricase as a thrifty gene is substantial and supports a role for evolutionary biology in the pathogenesis of the current obesity and diabetes epidemics.

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase.

The role of uric acid during primate evolution has remained elusive ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule in our serum. It has been difficult to generate a neutral or adaptive explanation in part because the uricase enzyme evolved to become a pseudogene in apes thus masking typical signals of sequence evolution. Adding to the difficulty is a lack of clarity on the functional role of uric acid in apes. One popular hypothesis proposes that uric acid is a potent antioxidant that increased in concentration to compensate for the lack of vitamin C synthesis in primate species ∼65 Ma. Here, we have expanded on our previous work with resurrected ancient uricase proteins to better resolve the reshaping of uricase enzymatic activity prior to ape evolution. Our results suggest that the pivotal death-knell to uricase activity occurred between 20 and 30 Ma despite small sequential modifications to its catalytic efficiency for the tens of millions of years since primates lost their ability to synthesize vitamin C, and thus the two appear uncorrelated. We also use this opportunity to demonstrate how molecular evolution can contribute to biomedicine by presenting ancient uricases to human immune cells that assay for innate reactivity against foreign antigens. A highly stable and highly catalytic ancient uricase is shown to elicit a lower immune response in more human haplotypes than other uricases currently in therapeutic development.

Combining Ancestral Reconstruction with Folding-Landscape Simulations to Engineer Heterologous Protein Expression.

Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations.

Brief report: The uricase mutation in humans increases our risk for cancer growth.

BACKGROUND: Recent studies suggest that fructose, as well as its metabolite, uric acid, have been associated with increased risk for both cancer incidence and growth. Both substances are known to cause oxidative stress to mitochondria and to reduce adenosine triphosphate (ATP) production by blocking aconitase in the Krebs cycle. The uricase mutation that occurred in the Miocene has been reported to increase serum uric acid and to amplify the effects of fructose to stimulate fat accumulation. Here we tested whether the uricase mutation can also stimulate tumor growth. METHODS: Experiments were performed in mice in which uricase was inactivated by either knocking out the gene or by inhibiting uricase with oxonic acid. We also studied mice transgenic for uricase. These mice were injected with breast cancer cells and followed for 4 weeks. RESULTS: The inhibition or knockout of uricase was associated with a remarkable increase in tumor growth and metastases. In contrast, transgenic uricase mice showed reduced tumor growth. CONCLUSION: A loss of uricase increases the risk for tumor growth. Prior studies have shown that the loss of the mutation facilitated the ability of fructose to increase fat which provided a survival advantage for our ancestors that came close to extinction from starvation in the mid Miocene. Today, however, excessive fructose intake is rampant and increasing our risk not only for obesity and metabolic syndrome, but also cancer. Obesity-associated cancer may be due, in part, to a mutation 15 million years ago that acted as a thrifty gene.

CRISPR-Cas9-mediated reactivation of the uricase pseudogene in human cells prevents acute hyperuricemia.

The utility of CRISPR-Cas9 to repair or reverse diseased states that arise from recent genetic mutations in the human genome is now widely appreciated. The use of CRISPR to "design" the outcomes of biology is challenged by both specialized ethicists and the general public. Less of a focus, however, is the ability of CRISPR to provide metabolic supplements or prophylactic molecules that improve long-term human health by overwriting ancient evolutionary events. Here, we use CRISPR to genomically integrate a functional uricase gene that encodes an enzymatically active protein into the human genome. These uricase-producing cells are able to reduce or even eliminate high concentrations of exogenous uric acid despite the enzyme being localized to peroxisomes. Our evolutionary engineered cells represent the first instance of the primate ape lineage expressing a functional uricase encoded in the genome within the last 20 million years. We anticipate that human cells expressing uricase will help prevent hyperuricemia (including gout) as well as hypertension and will help protect against fatty liver disease in the future.

Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction.

Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody-based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96-Padua as compared with AAV2/8-human FIX-Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.

Evolution of bacterial steroid biosynthesis and its impact on eukaryogenesis.

Steroids are components of the eukaryotic cellular membrane and have indispensable roles in the process of eukaryotic endocytosis by regulating membrane fluidity and permeability. In particular, steroids may have been a structural prerequisite for the acquisition of mitochondria via endocytosis during eukaryogenesis. While eukaryotes are inferred to have evolved from an archaeal lineage, there is little similarity between the eukaryotic and archaeal cellular membranes. As such, the evolution of eukaryotic cellular membranes has limited our understanding of eukaryogenesis. Despite evolving from archaea, the eukaryotic cellular membrane is essentially a fatty acid bacterial-type membrane, which implies a substantial bacterial contribution to the evolution of the eukaryotic cellular membrane. Here, we address the evolution of steroid biosynthesis in eukaryotes by combining ancestral sequence reconstruction and comprehensive phylogenetic analyses of steroid biosynthesis genes. Contrary to the traditional assumption that eukaryotic steroid biosynthesis evolved within eukaryotes, most steroid biosynthesis genes are inferred to be derived from bacteria. In particular, aerobic deltaproteobacteria (myxobacteria) seem to have mediated the transfer of key genes for steroid biosynthesis to eukaryotes. Analyses of resurrected steroid biosynthesis enzymes suggest that the steroid biosynthesis pathway in early eukaryotes may have been similar to the pathway seen in modern plants and algae. These resurrected proteins also experimentally demonstrate that molecular oxygen was required to establish the modern eukaryotic cellular membrane during eukaryogenesis. Our study provides unique insight into relationships between early eukaryotes and other bacteria in addition to the well-known endosymbiosis with alphaproteobacteria.

Molecular coevolution of coagulation factor VIII and von Willebrand factor.

Ancestral sequence reconstruction provides a unique platform for investigating the molecular evolution of single gene products and recently has shown success in engineering advanced biological therapeutics. To date, the coevolution of proteins within complexes and protein-protein interactions is mostly investigated in silico via proteomics and/or within single-celled systems. Herein, ancestral sequence reconstruction is used to investigate the molecular evolution of 2 proteins linked not only by stabilizing association in circulation but also by their independent roles within the primary and secondary hemostatic systems of mammals. Using sequence analysis and biochemical characterization of recombinant ancestral von Willebrand factor (VWF) and coagulation factor VIII (FVIII), we investigated the evolution of the essential macromolecular FVIII/VWF complex. Our data support the hypothesis that these coagulation proteins coevolved throughout mammalian diversification, maintaining strong binding affinities while modulating independent and distinct hemostatic activities in diverse lineages.

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase.

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Ancestral Sequence Reconstruction: From Chemical Paleogenetics to Maximum Likelihood Algorithms and Beyond.

As both a computational and an experimental endeavor, ancestral sequence reconstruction remains a timely and important technique. Modern approaches to conduct ancestral sequence reconstruction for proteins are built upon a conceptual framework from journal founder Emile Zuckerkandl. On top of this, work on maximum likelihood phylogenetics published in Journal of Molecular Evolution in 1996 was one of the first approaches for generating maximum likelihood ancestral sequences of proteins. From its computational history, future model development needs as well as potential applications in areas as diverse as computational systems biology, molecular community ecology, infectious disease therapeutics and other biomedical applications, and biotechnology are discussed. From its past in this journal, there is a bright future for ancestral sequence reconstruction in the field of evolutionary biology.

Stabilization of Near-Infrared Fluorescent Proteins by Packaging in Virus-like Particles.

Near-IR fluorescent Qß virus-like particles (VLPs) were produced in a high yield by packaging highly red-shifted monomeric and dimeric versions of biliverdin-dependent fluorescent proteins within the capsid shell. The simple addition of biliverdin hydrochloride to the medium during or after Escherichia coli protein expression was enough to produce fully matured encapsidated fluorophores. The packaged near-IR proteins exhibited identical photochemical properties to their nonencapsidated analogues but were far more stable toward heat, chaotrope-induced denaturation, and proteolysis. Noninvasive in vivo imaging showed the VLPs to traffic primarily to the liver after systemic injection in mice, revealing that the particles were easily detected by a standard instrument.

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding.

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

Incorporation of Modified Amino Acids by Engineered Elongation Factors with Expanded Substrate Capabilities.

Noncanonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, in vivo incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proofreading aa-tRNAs, which substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Here, we demonstrate an alternative strategy leveraging computational analysis to broaden EF-Tu's substrate specificity. Evolutionary analysis of EF-Tu and a naturally evolved specialized elongation factor, SelB, provide the opportunity to engineer EF-Tu by targeting amino acid residues that are associated with functional divergence between the two ancient paralogues. Employing amber codon suppression, in combination with mass spectrometry, we identified two EF-Tu variants with non-native substrate compatibility. Additionally, we present data showing these EF-Tu variants contribute to host organismal fitness, working cooperatively with components of native and engineered translation machinery. These results demonstrate the viability of our computational method and lend support to corresponding assumptions about molecular evolution. This work promotes enhanced polyspecific EF-Tu behavior as a viable strategy to expand ncAA scope and complements ongoing research emphasizing the importance of a comprehensive approach to further expand the genetic code.

On the Origin of Isoprenoid Biosynthesis.

Isoprenoids and their derivatives represent the largest group of organic compounds in nature and are distributed universally in the three domains of life. Isoprenoids are biosynthesized from isoprenyl diphosphate units, generated by two distinctive biosynthetic pathways: mevalonate pathway and methylerthritol 4-phosphate pathway. Archaea and eukaryotes exclusively have the former pathway, while most bacteria have the latter. Some bacteria, however, are known to possess the mevalonate pathway genes. Understanding the evolutionary history of these two isoprenoid biosynthesis pathways in each domain of life is critical since isoprenoids are so interweaved in the architecture of life that they would have had indispensable roles in the early evolution of life. Our study provides a detailed phylogenetic analysis of enzymes involved in the mevalonate pathway and sheds new light on its evolutionary history. The results suggest that a potential mevalonate pathway is present in the recently discovered superphylum Candidate Phyla Radiation (CPR), and further suggest a strong evolutionary relationship exists between archaea and CPR. Interestingly, CPR harbors the characteristics of both the bacterial-type and archaeal-type mevalonate pathways and may retain signatures regarding the ancestral isoprenoid biosynthesis pathway in the last universal common ancestor. Our study supports the ancient origin of the mevalonate pathway in the three domains of life as previously inferred, but concludes that the evolution of the mevalonate pathway was more complex.

Structural and Dynamics Comparison of Thermostability in Ancient, Modern, and Consensus Elongation Factor Tus.

Rationally engineering thermostability in proteins would create enzymes and receptors that function under harsh industrial applications. Several sequence-based approaches can generate thermostable variants of mesophilic proteins. To gain insight into the mechanisms by which proteins become more stable, we use structural and dynamic analyses to compare two popular approaches, ancestral sequence reconstruction (ASR) and the consensus method, used to generate thermostable variants of Elongation Factor Thermo-unstable (EF-Tu). We present crystal structures of ancestral and consensus EF-Tus, accompanied by molecular dynamics simulations aimed at probing the strategies employed to enhance thermostability. All proteins adopt crystal structures similar to extant EF-Tus, revealing no difference in average structure between the methods. Molecular dynamics reveals that ASR-generated sequences retain dynamic properties similar to extant, thermostable EF-Tu from Thermus aquaticus, while consensus EF-Tu dynamics differ from evolution-based sequences. This work highlights the advantage of ASR for engineering thermostability while preserving natural motions in multidomain proteins.

De novo active sites for resurrected Precambrian enzymes.

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.