Asymmetric Synthesis of α-Chloroamides via Photoenzymatic Hydroalkylation of Olefins.

Photoenzymatic intermolecular hydroalkylations of olefins are highly enantioselective for chiral centers formed during radical termination but poorly selective for centers set in the C-C bond-forming event. Here, we report the evolution of a flavin-dependent "ene"-reductase to catalyze the coupling of α,α-dichloroamides with alkenes to afford α-chloroamides in good yield with excellent chemo- and stereoselectivity. These products can serve as linchpins in the synthesis of pharmaceutically valuable motifs. Mechanistic studies indicate that radical formation occurs by exciting a charge-transfer complex templated by the protein. Precise control over the orientation of molecules within the charge-transfer complex potentially accounts for the observed stereoselectivity. The work expands the types of motifs that can be prepared using photoenzymatic catalysis.

A Bio-logical Approach to Catalysis in the Pharmaceutical Industry.

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.

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function.

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.

Structure of the cyanobactin oxidase ThcOx from Cyanothece sp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD.

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 Šat 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 Šdid 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.

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP.

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.

Accurate quantification of modified cyclic peptides without the need for authentic standards.

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 ICPMS, 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.

Derivatisable Cyanobactin Analogues: A Semisynthetic Approach.

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.

Structural analysis of leader peptide binding enables leader-free cyanobactin processing.

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 ß-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.

The structural biology of patellamide biosynthesis.

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.

The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster.

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.

An efficient method for the in vitro production of azol(in)e-based cyclic peptides.

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.

Identification of fluorinases from Streptomyces sp MA37, Norcardia brasiliensis, and Actinoplanes sp N902-109 by genome mining.

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.

Evaluation of an implantable MOSFET dosimeter designed for use with hypofractionated external beam treatments and its applications for breast and prostate treatments.

PURPOSE: An implantable metal-oxide semiconductor field effect transistors-based dosimeter has recently been developed for the in vivo monitoring of hypofractionated radiotherapy. This DVS-HFT dosimeter is designed for fraction sizes of 340-950 cGy and can also be used for bis in die fraction monitoring. The current work reports on the testing and evaluation of this dosimeter, including both its basic characteristics as well as its performance during simulated clinical treatment plans. METHODS: The authors tested the dose rate dependence of this dosimeter (300 MU/min versus 600 MU/min), the treatment time dependence (4 min per treatment versus up to 60 min per treatment), and the dose and energy dependence (6 and 18 MV irradiations of 700-900 cGy per fraction). Additionally, they irradiated the detectors in-phantom with breast and prostate hypofractionated treatments. RESULTS: The detectors showed no significant dose rate, treatment time, energy, or dose dependence. Furthermore, the detectors were found to perform within manufacturer tolerances for all hypofractionated treatments examined, accurately reporting the measured dose (average disagreement of - 0.65%). CONCLUSIONS: These dosimeters appear well suited for in vivo monitoring of hypofractionated radiotherapy doses, and thereby, have the potential to improve patient care.

Investigation of an implantable dosimeter for single-point water equivalent path length verification in proton therapy.

PURPOSE: In vivo range verification in proton therapy is highly desirable. A recent study suggested that it was feasible to use point dose measurement for in vivo beam range verification in proton therapy, provided that the spread-out Bragg peak dose distribution is delivered in a different and rather unconventional manner. In this work, the authors investigate the possibility of using a commercial implantable dosimeter with wireless reading for this particular application. METHODS: The traditional proton treatment technique delivers all the Bragg peaks required for a SOBP field in a single sequence, producing a constant dose plateau across the target volume. As a result, a point dose measurement anywhere in the target volume will produce the same value, thus providing no information regarding the water equivalent path length to the point of measurement. However, the same constant dose distribution can be achieved by splitting the field into a complementary pair of subfields, producing two oppositely "sloped" depth-dose distributions, respectively. The ratio between the two distributions can be a sensitive function of depth and measuring this ratio at a point inside the target volume can provide the water equivalent path length to the dosimeter location. Two types of field splits were used in the experiment, one achieved by the technique of beam current modulation and the other by manipulating the location and width of the beam pulse relative to the range modulator track. Eight MOSFET-based implantable dosimeters at four different depths in a water tank were used to measure the dose ratios for these field pairs. A method was developed to correct the effect of the well-known LET dependence of the MOSFET detectors on the depth-dose distributions using the columnar recombination model. The LET-corrected dose ratios were used to derive the water equivalent path lengths to the dosimeter locations to be compared to physical measurements. RESULTS: The implantable dosimeters measured the dose ratios with a reasonable relative uncertainty of 1%-3% at all depths, except when the ratio itself becomes very small. In total, 55% of the individual measurements reproduced the water equivalent path lengths to the dosimeters within 1 mm. For three dosimeters, the difference was consistently less than 1 mm. Half of the standard deviations over the repeated measurements were equal or less than 1 mm. CONCLUSIONS: With a single fitting parameter, the LET-correction method worked remarkably well for the MOSFET detectors. The overall results were very encouraging for a potential method of in vivo beam range verification with millimeter accuracy. This is sufficient accuracy to expand range of clinical applications in which the authors could use the distal fall off of the proton depth dose for tight margins.