Amino Acids, Peptides and Proteins Volume 38

By Etelka Farkas, Maxim Ryadnov

The Royal Society of Chemistry

Copyright © 2014 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-585-8

Contents

Preface Etelka Farkas and Maxim Ryadnov, v,
Oxidative transformations of amino acids and peptides catalysed by Cytochromes P450 Max J. Cryle, Clara Brieke and Kristina Haslinger, 1,
NMR investigation of disulfide containing peptides and proteins Katalin E. Kövér and Gyula Batta, 37,
Relaxin and its role in fibrotic diseases Linda Jiaying Chan, Chrishan S. Samuel, Frances Separovic, Mohammed Akhter Hossain and John D. Wade, 60,
Biofunctional peptide design Maxim G. Ryadnov, 79,
Self-assembly of designed peptides and their nanomaterials applications Hiroshi Tsutsumi and Hisakazu Mihara, 122,
Integrating chemical cross-linking with mass spectrometric analysis of peptides and proteins André Henrion, 151,
Peptidome analysis: tools and technologies Paulina D. Rakowska and Maxim G. Ryadnov, 172,
Peptide and protein based pharmaceuticals Gábor Mezo, 203,

CHAPTER 1

Oxidative transformations of amino acids and peptides catalysed by Cytochromes P450

Max J. Cryle, Clara Brieke and Kristina Haslinger DOI: 10.1039/9781849737081-00001

1 Introduction

1.1 Oxidation in nature

Enzymatic oxidation is an essential reaction in both primary and secondary metabolism. Owing to the large variety of substrates and types of reactions to be performed, a myriad of oxidoreductases has evolved (see Table 1). In general, the electron transfer is mediated by cofactors that together with the protein environment create specific redox potentials. These characteristic potentials determine the kind of reaction that can be catalysed by a certain class of enzymes. A very abundant and prominent class of oxidoreductases comprises Cytochrome P450 monooxygenases that will be subject of this book chapter in the context of oxidation of amino acids and peptides.

1.2 Cytochromes P450

1.2.1 Cytochrome P450 superfamily. In 1962 Cytochromes P450 (P450s) were described for the first time as coloured pigments in rat liver microsomes. As described in this publication P450s show a very characteristic absorption maximum at 450 nm when the protein solution is saturated with CO and subsequently reduced with dithionite. This maximum was later shown to be unique for cytochromes bearing a prosthetic, thiolate co-ordinated low-spin b-type haem. The thiolate ligand was subsequently identified as an invariant cysteine residue which lies within a highly conserved region of the protein. High sequence conservation in proximity to the haem and in other sections of the protein now allows the facile identification of new P450s based on predicted open reading frames. In this way more than eleven thousand genes through all kingdoms of life have been classified as members of the P450 superfamily and grouped into families and subfamilies based upon their sequence homologies.

1.2.2 Structure. The first X-ray structure of CYP101A1 (P450CAM; P450 nomenclature will be given along with the general enzyme name when available) revealed a characteristic P450 fold that has been shown to be well conserved amongst the superfamily (Fig. 1). The typical structure shows a high content of α-helices named in letters starting at the N-terminus (A to L) and a few β-strands. The most prominent helix is the I-helix spanning the whole molecule and coming in close proximity to the distal plane of the haem. The F and G helices, as the most mobile parts of the protein, are supposed to restrict the entry of potential substrates into the active site cavity. In the low spin, resting state the ferric haem (Fe(III)) is coordinated to a distal water molecule.

1.2.3 Catalytic cycle. In the catalytic cycle of P450CAM as depicted in Fig. 2, the entry of the substrate into the active site displaces the distal water molecule leading to high spin ferric haem (Fe(III)). This is observable as a shift in absorption in the visible spectrum (decrease of absorption at 420 nm and increase at 390 nm). Electron transfer from a redox partner leads to reduction of the haem iron followed by formation of a ferrous dioxy complex with molecular oxygen (Fe(II)+-O2). Sequential transfer of a second electron and a proton lead to formation of Compound 0 (Fe(III)+-OOH). After a second protonation, the O-O bond is cleaved and water is released, which leads to the formation of the reactive ferryl-oxo porphyrin cation radical, Compound I (Fe(IV)+-O). Following hydrogen atom abstraction from the substrate, radical recombination leads to the formation of the hydroxylated product which is released from the active site while the monooxygenase relaxes to its ferric resting state.

In vitro the proton and electron transfer steps can sometimes be cir cumvented by saturating the ferric haem with hydrogen peroxide, a process called the peroxide shunt pathway. The advantage of this technique is the simplification of the reaction as neither redox partners nor expensive cofactors (NADH or NADPH) are required.

1.2.5 Redox partners. The interplay of P450s with electron transfer systems is almost as diverse as the reactions catalysed by this machinery. Hannemann et al. grouped all known P450-redox systems into ten classes characterised in part by the number of polypeptides involved (one, two or three) and localisation of the system (cytosolic or ER membrane bound). The most well-studied P450, P450CAM, is the prototype of a bacterial member of class I. Electron transfer occurs between the 2Fe-2S iron-sulfur cluster of putidaredoxin (Pd) and the P450. An NADH-dependent FAD-containing ferredoxin reductase, putidaredoxin reductase (PdR), reduces the oxidised putidaredoxin after each electron transfer to the P450. Mitochondrial P450s share the same topology, but many systems are membrane-associated. Most liver-microsomal P450s, however, belong to class II, where the redox system is incorporated in a single polypeptide chain (diflavin reductases). Electrons are transferred from an FMN cofactor via an FAD cofactor to the P450. These two classes (I and II) are the most important ones and their members have been studied extensively.

Despite this, bacterial P450s are often found as "orphan" genes with no genes coding for potential redox partners in the surrounding regions. Biosynthetic gene clusters for glycopeptide antibiotics, for example, contain several genes coding for P450s but none for potential redox partners. In order to study these P450s the peroxide shunt pathway is applied and/or heterologous redox systems are tested. However, some P450s show strong specificity for their redox partners because of protein-protein interactions and the delicate regulation of redox potentials. In the P450CAM catalytic cycle putidaredoxin reductase (PdR) can be replaced by palustrisredoxin reductase (PuxR) whereas the same substitution on ferredoxin level doesn't allow electron transfer. For other P450s, however, the palustrisredoxin/ palustrisredoxin reductase (Pux/PuxR) system has successfully been employed as heterologous electron donors. This indicates the potential to exploit P450s for biocatalytic processes even where an endogenous redox system has not been identified.

1.2.5 Reactions. Cytochrome P450 enzymes play very important roles in all kingdoms of life. On the one hand they are involved in catabolic processes for the degradation of xenobiotics, such as pharmaceuticals, pesticides and other potentially detrimental compounds that are taken up by an organism. On the other hand P450s are key players in the biosynthesis of many secondary metabolites. In mammals these range from steroids and vitamins to fatty acids, whereas in prokaryotes many products are part of biological warfare between individual species (e.g. glycopeptide antibiotics produced by Actinomycetes). However, there are also examples of eukaryotes that take advantage of such metabolites in inter- and intraspecific competition. Many of these secondary metabolites are based on amino acids or peptides that undergo several modifications until they reach their full biological activity. The typical P450 catalysed reactions within this maturation process are hydroxylation, epoxidation, heteroatom oxidation or phenolic coupling. In some cases the substrates are free in solution whereas in other cases they are presented to the monooxygenase by a carrier protein.

1.3 Secondary metabolites from amino acids and peptides

A wide range of secondary metabolites in prokaryotes and eukaryotes are derived from amino acids and peptides. In particular, prokaryotic peptide derivatives have gained great importance as their antibiotic effects are exploited in clinical applications (penicillin, cephalosporin, vancomycin, bottromycin A2). These peptides can either be of ribosomal (e.g. lantibiotics) or of non-ribosomal origin (e.g. glycopeptide antibiotics).

1.3.1 Ribosomal peptides. Secondary metabolites derived from ribosomal peptides have a variety of appearances and functions. Their only common feature is that they are transcribed from genes and translated into a precursor peptide consisting of leader and core sequence. The leader sequence serves as a guide through maturation of the core peptide by recruiting tailoring enzymes and it mediates the secretion of the product (see Fig. 3). The most abundant post-translational modifications are listed in Table 2 and have been reviewed in detail by McIntosh et al.

The mechanism and order of these maturation steps strongly depend on the substance class. In the discussion we will focus on the maturation of lantibiotics which involves the actions of P450 enzymes.

All genes contributing to the biosynthesis of a certain lantibiotic are organised within a gene cluster starting with lanA, the gene coding for the precursor peptide. All tailoring enzymes such as the lanthionine synthetase are encoded downstream of the lanA gene. Lanthionine synthetase is a homomeric or heteromeric bifunctional complex that catalyses the dehydration of serine or threonine and the subsequent addition of the thiol group of a cysteine. This thioether forms the lanthionine bridges that provide extraordinary rigidity and stability against proteases to the peptides (see Fig. 7). It has been shown for lacticin 481 that basal synthetase activity can be detected in the absence of the leader whilst the directionality of processing from N- to C-terminus is lost. Furthermore, leader sequences were found to direct synthetase activity even in trans but with decreased efficiency compared to the intact precursor peptide. Following the actions of lanthionine synthetase and other tailoring enzymes, the leader sequence mediates the recognition by cellular secretion systems and is cleaved from the mature peptide. Most lantibiotics have been shown to be inactive in presence of the leader. Thus, the leader peptide not only drives peptide maturation, it also takes part in secretion and self-immunity.

From a commercial point of view ribosomal peptides are of significant interest as they are easily modified to generate a range of new bioactive compounds. Simple exchange of codons in the core sequence can afford fully functional or even improved compounds as long as the leader remained unchanged. Furthermore, a new application utilising the versatile tool box of naturally occurring post-translational modifications mediated by leader peptides called leader peptide assisted biosynthesis (LPB) has been established. Target peptides can be expressed in fusion with a helper leader in order to improve peptide characteristics such as pharmacokinetics, bioavailability, stability, or conformational uniformity amongst others. LBP can even be combined with the use of commercially available proteases, ensuring full control over the complete maturation and facilitating isolation of the final peptide.

1.3.2 Non-ribosomal peptides. The other group of peptidic secondary metabolites is of non-ribosomal origin. These peptides are assembled by large enzyme complexes called Non-Ribosomal Peptide Synthetases (NRPS). The generic topology of an NRPS is characterised by a modular organisation where each module specifically incorporates a certain amino acid into the peptide (see Fig. 4). The modules are arranged in one or more polypeptide chains with the exact number of modules depending on the system. The minimal module consists of an Adenylation (A), a Peptidyl Carrier (P or PCP) and a Condensation (C) domain. These modules can be extended by further domains such as Epimerisation (E), Methyltransferase (Mt), Cyclisation (Cy), Oxidation (O), Reduction (R) or Thioesterase (Te) domains. In the catalytic process the A-domain activates an amino acid by forming an acyl adenylate with ATP and transfers it onto the 40-phosphopantetheinyl (Ppant) arm of the PCP domain, where it forms a reactive thioester. The Ppant group shuttles amino acids from the active site of the A-domain to the active site of the C-domain. The C-domain bears two cavities where the amino acids (i) and (i + 1) can be accommodated. In these positions the free N-terminus of the tethered (i + 1) amino acid attacks the Ppant-bound C-terminus of residue (i) leading to peptide bond formation. The nascent peptide chain remains attached to the (i + 1) Ppant and is transferred to the active site of the next condensation domain (see Fig. 4). The final module of the NRPS contains an additional thioesterase domain that cleaves the thioester bond between the last PCP and the peptide. Depending on the system this step yields either a linear or a N-C macrocyclic peptide.

The number of additional domains in these modules (E, Mt, Cy etc.) already suggests that with this system peptides with a range of unusual amino acids (methylated, Mt-domain; cyclised, Cy-domain) and amino acid configurations (E-domain) can be produced. Further diversity is introduced by incorporation of non-proteogenic amino acids like hydroxyphenylglycine. An array of tailoring enzymes such as methyltransferases, halogenases and oxygenases modify the peptide backbone to obtain highly stable and bioactive compounds. Specific transferases attach sugar or fatty acid decorations depending on subcellular localisation and other required features as e.g. membrane permeability.

It was shown that the actions of A-domains are highly specific for the side chains of their substrates whereas C-domains are very specific towards the stereochemistry of amino acids. These mechanisms ensure the correct sequence and stereochemistry of the generated peptide. These characteristics can be exploited for the analysis of newly discovered NRPSs; new NRPS gene clusters are identified by the high homology of A-domains (up to 56% sequence similarity) and from the active site residues of A- and C-domains one can predict the sequence and stereochemistry of the product peptide with bioinformatic tools.

The modularity of the NRPS offers a vast platform for biotechnological applications in order to generate new peptides. The obvious approach, namely genetic swapping of domains or whole modules, was soon proven to be difficult: only a few examples afforded functional peptides in acceptable yields. Possible explanations are the high substrate specificities of the later synthetic domains, the inactivity of swapped domains due to incorrect boundary selection or inadequate communication between the domains and modules. Hahn and Stachelhaus identified short regions (15-25 amino acids) at the edges of NRPS subunits called communication-mediating domains (COM). These domains are crucial for the assembly of the NRPS and the communication between the subunits and thereby impose additional complications on domain swapping.

Eppelmann et al. have managed to alter the substrate specificity of A-domains by site-directed mutagenesis. However, this approach is only suitable for conservative changes, where few mutations are needed and the substrate is still tolerated by the downstream domains.

Ribosomal and non-ribosomal peptides are processed by a myriad of tailoring enzymes contributing to the rich structural and functional diversity of these secondary metabolites. One important modification is oxidation, e.g. oxidation of thiazolines, oxidative coupling of aromatic side chains, β-hydroxylations and epoxidations. These reactions can be catalysed by members of the P450 superfamily on amino acid and peptide level and will now be discussed.

2 P450-catalysed oxidative modification of peptides and amino acids

2.1 Hydroxylation

P450-catalysed hydroxylation of amino acids and peptides is mainly found in secondary metabolic pathways. Gene cluster analyses have identified a large number of P450s implicated in such reactions, although in many cases only homology studies have been used for assigning function and still less is known about the exact substrates. In the biosynthesis of natural products derived from amino acids P450s often act as tailoring enzymes on PCP-bound substrates. This is especially the case for the β-hydroxylation of amino acids, which will be discussed below.

2.1.1 β-hydroxylation of PCP-bound amino acids. Hydroxylated amino acids are widely used intermediates in the biosynthesis of a variety of complex secondary metabolites. Many substances with antibiotic, antiviral or antifungal properties are among these natural products and are highly interesting for biomedical research. Somewhat surprisingly, most P450-catalysed oxidations on the b-position of amino acids are performed on carrier protein-bound substrates. The desired amino acid is recognised by the A-domain of an NRPS and loaded onto the PCP domain. The aminoacyl-PCP then serves as a substrate for a P450, which catalyses the oxidation of the b-position of the tethered amino acid: this reaction often proceeds with high stereoselectivity. Following this oxidation, the hydroxylated amino acid is released by a thioesterase domain (see Fig. 5) and further metabolised. β-Hydroxylation of tyrosine, tryptophan, histidine and valine residues have been found to proceed via this pathway and it is hypothesised that restricting oxidation to amino acids in their PCP-bound form is used to control the fraction of amino acids diverted into secondary metabolism.

(Continues...)Excerpted from Amino Acids, Peptides and Proteins Volume 38 by Etelka Farkas, Maxim Ryadnov. Copyright © 2014 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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