Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily.

Insight regarding how diverse enzymatic functions and reactions have evolved from ancestral scaffolds is fundamental to understanding chemical and evolutionary biology, and for the exploitation of enzymes for biotechnology. We undertook an extensive computational analysis using a unique and comprehensive combination of tools that include large-scale phylogenetic reconstruction to determine the sequence, structural, and functional relationships of the functionally diverse flavin mononucleotide-dependent nitroreductase (NTR) superfamily (>24,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions). Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a minimal flavin-binding scaffold. We identified the structural design principle for this divergence: Insertions at key positions in the minimal scaffold that, combined with the fixation of key residues, have led to functional specialization. These results will aid future efforts to delineate the emergence of functional diversity in enzyme superfamilies, provide clues for functional inference for superfamily members of unknown function, and facilitate rational redesign of the NTR scaffold

Thrice upon a time: The repeated emergence of a novel enzymatic function from an evolvable protein scaffold

Understanding the emergence of new protein functions from their ancestors is a long-standing challenge in biology and biotechnology; many questions remain unanswered. How can one protein scaffold support multiple distinct functions? How are diverse functions of a superfamily connected? How are major functional switches achieved? Large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies have now begun to provide comprehensive views of functional diversity and evolutionary relationships. Intriguing insights can be gained: promiscuous activities are prevalent and many divergent proteins retain functional connectivity via enzyme promiscuity1.Interested in the varied biological and biotechnological roles of FMN-dependent “nitroreductase” enzymes (NTRs), we undertook extensive computational and functional analyses to determine sequence, structural and functional relationships2. This large and diverse superfamily contains \u3e80,000 sequences from all domains of life, 54 structures, and \u3e10 enzymatic functions. Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a highly “evolvable” minimal flavin-binding scaffold. To investigate the diverse NTR sequence space for the capacity to catalyze nitroreduction, we synthesized \u3e500 genes and performed high-throughput activity screening to profile 18 in vivo substrates. In vitro kinetic analysis was subsequently performed on 112 enzymes against 32 substrates (vs. 2 nicotinamide cofactors), equating to \u3e7,000 reactions3. We demonstrated that only four of the 22 distinct superfamily subgroups display canonical nitroaromatic reductase activities. Eight additional subgroups display occasional promiscuous activities with selected substrates, and 10 subgroups display no nitroreductase activity. Structural analyses revealed the underlying molecular details: nitroreduction has emerged three distinct times in the superfamily via three unique molecular solutions - loop insertions at three different positions in the NTR scaffold, combined with the fixation of key residues, have independently led to functional specialization. These results are now facilitating the rational redesign of the NTR scaffold. Our work provides clues for functional inference for sequences of unknown function, and will aid future efforts to exploit evolvable scaffolds for engineering, and understand the emergence of functional diversity in enzyme superfamilies. Baier F, Copp JN, Tokuriki N. Biochemistry. 2016 Nov 22;55(46):6375-6388. Akiva E*, Copp JN*, Tokuriki N, Babbitt PC. Proc Natl Acad Sci U S A. 2017 114(45):E9549-E9558. Copp JN, Morales DM, Chang S, Jiang K, Akiva E, Babbitt PC, Tokuriki N. in prep

Exploring sequence-function space in the Old Yellow Enzyme superfamily

Biotechnology and bioinformatics have made it increasingly apparent that there is a vast wealth of protein ‘dark matter’, i.e., sequence and functional information that is yet to be discovered and harnessed for fundamental or applied gains. For example, the superfamily of Old Yellow Enzymes (OYEs) with ~88 characterized enzymes in the literature, is shockingly underexplored, despite \u3e85 years of research and their proven industrial application. We have applied large scale bioinformatic and synthetic biology approaches to systematically sample and functionally characterize \u3e120 representatives across the entire OYE superfamily, which is comprised of \u3e70,000 members. Our efforts have more than doubled the current OYE knowledgebase and have yielded native biocatalysts with improved activity and expanded substrate specificity. Furthermore, our multidisciplinary approach serves as an adaptable pipeline for the analysis of other superfamilies, improving the current standard of investigative processes for the field. The comprehensive characterization of enzyme superfamilies, especially those with proven biocatalysis capabilities, offers tremendous opportunities for future developments of green and sustainable chemical processes

Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

A revised biosynthetic pathway for the cofactor F-420 in prokaryotes

Cofactor F420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho-L-lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho-L-lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F420-0. The C-terminal domain of FbiB then utilizes FMNH2 to reduce dehydro-F420-0, which produces mature F420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F420 from a recombinant F420 biosynthetic pathway in Escherichia coli

Metagenomics and sequence similarity networks expose cryptic sequence space to enable enzyme discovery and enhance engineering strategies

Biotechnology is dependent upon the extraordinary efficiency, specificity, and versatility of enzyme function. Over the last decade, the revolution in sequencing technologies has produced vast amounts of sequence information from diverse biological sources. However, we have few functional details about the majority of this data, and therefore have only harnessed a minute fraction of the repertoire of enzymes and metabolic pathways available in Nature. Strategies to predict and characterize the functions of unexplored sequence space are urgently needed. Here, we present an innovative approach to characterize and classify sequence, structure, and functional diversity of a diverse group of enzymes - the FMN-dependent nitroreductase superfamily. This superfamily is comprised of biotechnologically important enzymes1, yet only a small number of enzymes have been characterized. We undertook a comprehensive analysis, using a unique combination of sequence, structural, functional and phylogenetic characterizations (\u3e24,000 sequences, 54 structures and \u3e10 enzymatic functions) to create the first global view of the nitroreductase superfamily2 – of particular interest for biomedical, bioremediation, and biocatalysis applications. The superfamily was delineated into 22 distinct subgroups, 8 of which have no currently known function. Furthermore, we identified three “hot spots” within the nitroreductase scaffold that form the structural basis for the evolution of function, and revealed the key functional residues that have led to evolutionary adaptation through active site profiling. This information is instrumental to the rational redesign of the nitroreductase scaffold. We applied our new knowledge of the nitroreductase superfamily to screen \u3e7,000 metagenomes from public and private repositories to expose the true diversity of NTR enzymes, this approach resulted in an extensive final dataset of ~1M novel nitroreductases. Prominent and subgroup specific enrichment profiles for distinct metagenomic environments were also revealed by subgroup profiling. To further investigate this newly discovered sequence space, we are performing large scale enzymatic activity profiling (\u3e400 enzymes) to provide functional data on a vast number of novel nitroreductase enzymes, and develop an innovative “nitroreductase toolbox”, with wide-ranging potential for biotechnological applications. Roldan et al., FEMS Microbiol Rev 32, 474–500 (2008). Akiva, Copp et al., submitted

Catalysed activation of cyanobacterial biosynthetic pathways by phosphopantetheinyl transferases

Cyanobacteria produce a diverse array of natural products with significant potential inmany biotechnological, clinical and commercial applications. These includepharmaceuticals, such as antitumour products, antibiotics, immunosuppressants, anticholesterolemicsand anti-parasitic agents, as well as veterinary therapies andagrochemicals. These compounds are synthesised by complex secondary metabolismpathways involving polyketide synthases (PKS) and non-ribosomal peptide synthetases(NRPS), both of which require an essential phosphopantetheinyl transferase (PPT) fortheir activity.PPTs activate the acyl, aryl and peptidyl carrier proteins within various biosyntheticpathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue.Phylogenetic analysis of the large superfamily of PPTs has revealed two separatefamilies based on substrate specificity, which have been designated AcpS and Sfp-like.The AcpS PPT family activate acyl carrier proteins of fatty acid synthesis, while theSfp-like PPT family, typified by the Bacillus subtilis PPT Sfp, has diverse roles inprimary and secondary metabolism.The majority of cyanobacterial PPTs are of the Sfp-like PPT family and occur ingenomes lacking an AcpS PPT. Phylogenetic analysis uncovered a distinct clade ofcyanobacterial PPTs involved in heterocyst differentiation. Heterologous expressionand functional analysis of NsPPT, the heterocyst-associated PPT in Nodulariaspumigena NSOR10, represented the first characterisation of a cyanobacterial PPT.PCR-based screening was utilised to identify NsPPT and Southern hybridisationsuggested this was the only PPT encoded by the N.spumigena NSOR10 genome.Enzymatic analyses demonstrated the ability of NsPPT to phosphopantetheinylate PKSand NRPS carrier proteins from a range of metabolism pathways and cyanobacterialspecies.Nostoc punctiforme ATCC 29133 encodes three PPTs. One of these PPTs, NgcS, is alsoa heterocyst-associated PPT and is homologous to NsPPT of N.spumigena NSOR10.Expression and enzymatic analysis of NgcS from N. punctiforme ATCC 29133,revealed contrasting phosphopantetheinylation activity to that seen for NsPPT, andindicated that NgcS may have evolved to have a strict specificity for the glycolipidbiosynthesis pathway.Although the Sfp-like family of PPTs are normally associated with secondarymetabolite biosynthesis, Synechocystis sp. PCC 6803 harbours a unique Sfp-like PPT(Sppt) but does not produce NRPS or PKS compounds. Genetic disruption of Sppt wasattempted and expression of Sppt allowed the characterisation of its enzyme kinetics.Sppt displayed the ability to activate non-cognate cyanobacterial carrier proteins fromNRPS and PKS biosynthetic pathways, although only at a low level of activity. Thissuggested that wild-type Synechocystis sp. PCC6803 would not be suitable forheterologous expression of cyanobacterial secondary metabolites.These results have important implications regarding the expression and manipulation ofcyanobacterial bioactive compounds in heterologous hosts. Applications of this researchmay provide a biotechnological platform for the sustainable production ofcyanobacterial natural products

The Synechocystis sp. PCC6803 Sfp-type phosphopantetheinyl transferase does not possess characteristic broad-range activity

The cyanobacterium Synechocystis sp. PCC6803 harbours one phosphopantetheinyl transferase (PPTase), Sppt. Protein modelling supported previous bioinformatics analyses, which suggested that Sppt is a Sfp-type PPTase with the potential to phosphopantetheinylate a broad range of carrier proteins from both primary and secondary metabolism. However, no natural products are synthesised by this species, which raises interesting evolutionary and functional questions. Phosphopantetheinylation assays and kinetic data demonstrate that Sppt was able to activate its cognate fatty acid synthesis carrier protein, SACP, but was unable to effectively activate various cyanobacterial carrier proteins from secondary metabolism or glycolipid biosynthesis pathways. To our knowledge, this is the first example of a PPTase with a Sfp-type structure, but with activity more closely resembling AcpS-type enzymes. The broad-range PPTase from Nodularia spumigena NSOR10 was introduced into Synechocystis sp. PCC6803 and was shown to activate a noncognate carrier protein, in vivo. This engineered strain could provide a future biotechnological platform for the heterologous expression of cyanobacterial biosynthetic gene clusters

Revealing Unexplored Sequence-Function Space Using Sequence Similarity Networks

The rapidly expanding number of protein sequences found in public databases can improve our understanding of how protein functions evolve. However, our current knowledge of protein function likely represents a small fraction of the diverse repertoire that exists in nature. Integrative computational methods can facilitate the discovery of new protein functions and enzymatic reactions through the observation and investigation of the complex sequence-structure–function relationships within protein superfamilies. Here, we highlight the use of sequence similarity networks (SSNs) to identify previously unexplored sequence and function space. We exemplify this approach using the nitroreductase (NTR) superfamily. We demonstrate that SSN investigations can provide a rapid and effective means to classify groups of proteins, therefore exposing experimentally unexplored sequences that may exhibit novel functionality. Integration of such approaches with systematic experimental characterization will expand our understanding of the functional diversity of enzymes and their associated physiological roles