Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Analyzing the natural evolution of proteins by ancestral sequence reconstruction (ASR) can provide valuable information about the changes in sequence and structure that drive the development of novel protein functions. However, ASR has also been used as a protein engineering tool, as it often generates thermostable proteins which can serve as robust and evolvable templates for enzyme engineering. Importantly, ASR has the potential to provide an insight into the history of insertions and deletions that have occurred in the evolution of a protein family. Indels are strongly associated with functional change during enzyme evolution and represent a largely unexplored source of genetic diversity for designing proteins with novel or improved properties. Current ASR methods differ in the way they handle indels; inclusion or exclusion of indels is often managed subjectively, based on assumptions the user makes about the likelihood of each recombination event, yet most currently available ASR tools provide limited, if any, opportunities for evaluating indel placement in a reconstructed sequence. Graphical Representation of Ancestral Sequence Predictions (GRASP) is an ASR tool that maps indel evolution throughout a reconstruction and enables the evaluation of indel variants. This chapter provides a general protocol for performing a reconstruction using GRASP and using the results to create indel variants. The method addresses protein template selection, sequence curation, alignment refinement, tree building, ancestor reconstruction, evaluation of indel variants and approaches to library development.

NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

SeqScrub: a web tool for automatic cleaning and annotation of FASTA file headers for bioinformatic applications.

Data consistency is necessary for effective bioinformatic analysis. SeqScrub is a web tool that parses and maintains consistent information about protein and DNA sequences in FASTA file format, checks if records are current, and adds taxonomic information by matching identifiers against entries in authoritative biological sequence databases. SeqScrub provides a powerful, yet simple workflow for managing, enriching and exchanging data, which is crucial to establish a record of provenance for sequences found from broad and varied searches; for example, using BLAST on continually updated genome sequence sets. Headers standardized using SeqScrub can be parsed by a majority of bioinformatic tools, stay uniformly named between collaborators and contain informative labels to aid management of reproducible, scientific data. SeqScrub is available at http://bioinf.scmb.uq.edu.au/seqscrub.

The GMC superfamily of oxidoreductases revisited: analysis and evolution of fungal GMC oxidoreductases.

BACKGROUND: The glucose-methanol-choline (GMC) superfamily is a large and functionally diverse family of oxidoreductases that share a common structural fold. Fungal members of this superfamily that are characterised and relevant for lignocellulose degradation include aryl-alcohol oxidoreductase, alcohol oxidase, cellobiose dehydrogenase, glucose oxidase, glucose dehydrogenase, pyranose dehydrogenase, and pyranose oxidase, which together form family AA3 of the auxiliary activities in the CAZy database of carbohydrate-active enzymes. Overall, little is known about the extant sequence space of these GMC oxidoreductases and their phylogenetic relations. Although some individual forms are well characterised, it is still unclear how they compare in respect of the complete enzyme class and, therefore, also how generalizable are their characteristics. RESULTS: To improve the understanding of the GMC superfamily as a whole, we used sequence similarity networks to cluster large numbers of fungal GMC sequences and annotate them according to functionality. Subsequently, different members of the GMC superfamily were analysed in detail with regard to their sequences and phylogeny. This allowed us to define the currently characterised sequence space and show that complete clades of some enzymes have not been studied in any detail to date. Finally, we interpret our results from an evolutionary perspective, where we could show, for example, that pyranose dehydrogenase evolved from aryl-alcohol oxidoreductase after a change in substrate specificity and that the cytochrome domain of cellobiose dehydrogenase was regularly lost during evolution. CONCLUSIONS: This study offers new insights into the sequence variation and phylogenetic relationships of fungal GMC/AA3 sequences. Certain clades of these GMC enzymes identified in our phylogenetic analyses are completely uncharacterised to date, and might include enzyme activities of varying specificities and/or activities that are hitherto unstudied.

Structure and Function of the TIR Domain from the Grape NLR Protein RPV1.

The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices ("AE" interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.