Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling.

Background: The inability of biologics to pass the plasma membrane prevents their development as therapeutics for intracellular targets. To address the lack of methods for cytosolic protein delivery, we used the type III secretion system (T3SS) of Y. enterocolitica, which naturally injects bacterial proteins into eukaryotic host cells, to deliver monobody proteins into cancer cells.

Identification of a Novel Transasparaginase Activity of (bTG) for Sequence-Specific Bioconjugation.

The ability of 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.

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.

A Bio- 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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.