The ability of Bacillus subtilis transglutaminase (bTG) to functionalize BSA has been investigated using peptide mapping experiments. Interestingly, the conjugation was not detected on a glutamine but on an asparagine residue. A sequence determination study was further performed, and a sequence of 10 amino acids for site-specific conjugation was identified. A monobody showing no native reactivity with the bTG enzyme was produced with the identified peptide sequences and successfully conjugated to various types of substrates in very high yields (>90%) with a 1/1/1.5 ratio of protein/amine/enzyme. Direct conjugation to the amino linker of a small interfering RNA (siRNA) was achieved in good yield, and no impact on the siRNA activity was observed following the conjugation. The identified sequences were further engineered in VHH and IgG scaffolds, and successful conjugation could also be observed with both small molecules and siRNA, confirming the potential of bTG for site-specific enzymatic bioconjugation.

With renewed interest in therapeutic oligonucleotides, and at the same time the pharmaceutical industry set itself ambitious sustainability goals, there is a strong interest to investigate next-generation synthesis methods for these highly modified oligonucleotides. We herein report an RNA ligase catalyzed assembly of multiple, short oligonucleotides in aqueous solution to generate an siRNA product. A conversion of the non-native substrates to the desired product of 40 – 80 % was achieved.

Enzymes have the potential to catalyse complex chemical reactions with unprecedented selectivity, under mild conditions in aqueous media. Accordingly, there is serious interest from the pharmaceutical industry to utilize enzymes as biocatalysts to produce medicines in an environmentally sustainable and economic manner. Prominent advances in the field of biotechnology have transformed this potential into a reality. Using modern protein engineering techniques, in a matter of months it is possible to evolve an enzyme, which fits the demands of a chemical process, or even to catalyse entirely novel chemistry. Consequently, biocatalysis is routinely applied throughout the pharmaceutical industry for a variety of applications, ranging from the manufacture of large volumes of high value blockbuster drugs to expanding the chemical space available for drug discovery.

With advances in sequencing technology, uncharacterized proteins and domains of unknown function (DUFs) are rapidly accumulating in sequence databases and offer an opportunity to discover new protein chemistry and reaction mechanisms. The focus of this review, the formerly enigmatic YcaO superfamily (DUF181), has been found to catalyze a unique phosphorylation of a ribosomal peptide backbone amide upon attack by different nucleophiles. Established nucleophiles are the side chains of Cys, Ser, and Thr which gives rise to azoline/azole biosynthesis in ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products. However, much remains unknown about the potential for YcaO proteins to collaborate with other nucleophiles. Recent work suggests potential in forming thioamides, macroamidines, and possibly additional post-translational modifications. This review covers all knowledge through mid-2016 regarding the biosynthetic gene clusters (BGCs), natural products, functions, mechanisms, and applications of YcaO proteins and outlines likely future research directions for this protein superfamily.

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N-terminal "leader" sequence. We report herein the structural and biochemical characterization of BotP, a leucyl-aminopeptidase-like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro- mycin.

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Å at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content.

There is a growing interest in the use of cyclic peptides as therapeutics, but their efficient production is often the bottleneck in taking them forward in the development pipeline. We have recently developed a method to synthesise azole-containing cyclic peptides using enzymes derived from different cyanobactin biosynthetic pathways. Accurate quantification is crucial for calculation of the reaction yield and for the downstream biological testing of the products. In this study, we demonstrate the development and validation of two methods to accurately quantify these compounds in the reaction mixture and after purification. The first method involves the use of a HPLC coupled in parallel to an ESMS and an ICP-MS, hence correlating the calculated sulfur content to the amount of cyclic peptide. The second method is an NMR ERETIC method for quantifying the solution concentration of cyclic peptides. These methods make the quantification of new compounds much easier as there is no need for the use of authentic standards when they are not available.

Structural and biochemical analysis of the heterocyclase that acts on a ribosomally synthesized and post-translationally modified peptide identifies the basis for leader peptide activation and facilitates engineering of a constitutively active enzyme. Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.

Many natural cyclic peptides have potent and potentially useful biological activities. Their use as therapeutic starting points is often limited by the quantities available, the lack of known biological targets and the practical limits on diversification to fine-tune their properties. We report the use of enzymes from the cyanobactin family to heterocyclise and macrocyclise chemically synthesised substrates so as to allow larger-scale syntheses and better control over derivatisation. We have made cyclic peptides containing orthogonal reactive groups, azide or dehydroalanine, that allow chemical diversification, including the use of fluorescent labels that can help in target identification. We show that the enzymes are compatible and efficient with such unnatural substrates. The combination of chemical synthesis and enzymatic transformation could help renew interest in investigating natural cyclic peptides with biological activity, as well as their unnatural analogues, as therapeutics.

The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and l-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87% identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.

Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological `toolkit' to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized. The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain proteins PatA and PatG. The domain sequence is found in all cyanobactin-biosynthetic pathways characterized to date, implying a functional role in cyanobactin biosynthesis. Here, the crystal structure of the PatG DUF domain is reported and its binding interactions with plausible substrates are investigated.

The biosynthetic pathways for patellamide and related natural products have recently been studied by structural biology. These pathways produce molecules that have a complex framework and exhibit a diverse array of activity due to the variability of the amino acids that are found in them. As these molecules are difficult to synthesize chemically, exploitation of their properties has been modest. The patellamide pathway involves amino acid heterocyclization, peptide cleavage, peptide macrocyclization, heterocycle oxidation and epimerization; closely related products are also prenylated. Enzyme activities have been identified for all these transformations except epimerization, which may be spontaneous. This review highlights the recent structural and mechanistic work on amino acid heterocyclization, peptide cleavage and peptide macrocyclization. This work should help in using the enzymes to produce novel analogs of the natural products enabling an exploitation of their properties.

Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6-9 residues representing 11 out of the 20 canonical amino acids.

This work was funded by the Leverhulme Trust (RPG-2012–504), BBSRC (BB/K015508/1) and the University of St Andrews infrastructure is supported by a Wellcome Trust Capital Award (086036).