Clarissa M. Czekster

Kinetic Landscape of a Peptide Bond-Forming Prolyl Oligopeptidase

Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases.

Mechanisms of cyanobactin biosynthesis

Cyanobactins are a diverse collection of natural products that originate from short peptides made on a ribosome. The amino acids are modified in a series of transformations catalyzed by multiple enzymes. The patellamide pathway is the most well studied and characterized example. Here we review the structures and mechanisms of the enzymes that cleave peptide bonds, macrocyclise peptides, heterocyclise cysteine (as well as threonine and serine) residues, oxidize five-membered heterocycles and attach prenyl groups. Some enzymes operate by novel mechanisms which is of interest and in addition the enzymes uncouple recognition from catalysis. The normally tight relationship between these factors hinders biotechnology. The cyanobactin pathway may be particularly suitable for exploitation, with progress observed with in vivo and in vitro approaches.

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable, and capable of modulating protein–protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.Cyclic peptide macrocycles are promising anti-cancer and antimicrobial molecules. Here, the authors characterize the structure and catalytic mechanism of the prolyl oligopeptidase B from Basidiomycete fungi, showing that its dual macrocyclase-peptidase activity is crucial for amatoxin macrocyclization.

The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family

Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99-880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD, that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC-MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi.

Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1’s potential in biocatalysis.

CHAPTER 2:The Biosynthesis of Cyclic Peptides – RiPPs – An Overview

Cyclic peptides are of considerable interest because they possess protease resistant, rigid scaffolds that can be almost infinitely diversified. Their size and molecular complexity means that they are able to target protein–protein interactions, a task that current small molecule drugs struggle to achieve. Macrocyclic peptides can be synthesized using non-ribosomal peptide synthesis machineries – NRPS for short (see next chapter) – or through extensive modification of ribosomally synthesized peptide precursors (ribosomally synthesized and post-translationally modified peptides – RiPPs). RiPPs are attractive because they are genetically encoded and can be easily diversified. The same peptide precursors can be utilized to generate a wide array of natural products by “mixing-and-matching” enzymes involved in their post-translational modification. Here, we discuss the biosynthetic machineries producing the main classes of cyclic RiPPs.