Introduction

Natural products derived from microbial, plant or animal organisms constitute the largest source for medicinal drugs, either in unmodified form or as chemically modified derivatives (Newman and Cragg 2020). Mining of microbial genome sequences has strongly accelerated the elucidation process of biosynthetic pathways of known metabolites and revealed the presence of a large number of unknown biosynthetic gene clusters (BGCs). They are responsible for new extraordinary enzymes and an astonishing variety of secondary metabolites. As these BGCs are usually silent and their original triggers are diverse, unspecific or even hazardous, often only a targeted activation either in the natural host or in optimised expression hosts provides insights into their functions. The characterised secondary metabolite enzymes are able to catalyse stereoselective, stereospecific, efficient and energetically disfavoured reactions.

Cyclodipeptides (CDPs) are the smallest possible cyclic peptides from two amino acids with two peptide bonds. In nature, CDPs are assembled by either nonribosomal peptide synthetases (NRPSs) mostly in fungi or by cyclodipeptide synthases (CDPSs) mainly in bacteria. These two enzyme families differ not only in protein size and sequences but also in substrates and reaction mechanisms. NRPSs are large multi-modular proteins using free amino acids as substrates (Izoré and Cryle 2018; Payne et al. 2017). Diketopiperazine (DKP)-forming NRPSs are dimodular enzymes with typical peptide chain lengths of about 2300–2500 amino acids (Xu et al. 2014). In comparison, CDPSs consist only of 200–300 amino acids and hijack the activated aminoacyl-tRNAs from the ribosomal machinery for CDP formation (Fig. 1) (Gondry et al. 2009, 2018; Moutiez et al. 2017).

Fig. 1
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Cyclodipeptide formation catalysed by cyclodipeptide synthases with aminoacyl-tRNAs as substrates

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Modification of CDPs by tailoring enzymes

The DKP ring of the CDPs has an increased stability against proteolysis, in comparison to acyclic dipeptides (Borthwick 2012), making it a stable scaffold for diverse modifications by tailoring enzymes such as oxidoreductases including 2-oxoglutarate-dependent monooxygenases, cyclodipeptide oxidases (CDOs) and cytochrome P450 enzymes (P450s), methyltransferases (MTs) and prenyltransferases (Borgman et al. 2019; Canu et al. 2020). Known CDP modification reactions by CDPS-associated tailoring enzymes are listed in Table 1. The products of CDPS-related pathways exhibit diverse pharmacological effects such as antibiotic (Cain et al. 2003), antifungal (Musetti et al. 2007; Ström et al. 2002), anti-inflammatory (Minelli et al. 2012), immunosuppressive (Waring and Beaver 1996) and antitumor activities (Yamazaki et al. 2012). Bicyclomycin, for example, is used as an antibiotic for treatment of traveller’s diarrhoea caused by gram-negative bacteria such as E. coli, Klebsiella, Shigella or Salmonella species. It has a unique pharmacological mechanism by selectively inhibiting the bacterial transcription termination factor Rho and shows synergetic effects with other antibiotics (Kohn and Widger 2005; Lawson et al. 2016). Mycocyclosin is essential for the viability of Mycobacterium tuberculosis. The P450 enzyme CYP121 involved in its formation could be therefore considered an alternative target for potential drugs in the treatment of tuberculosis, which is still responsible for 1.5 million deaths worldwide per year (Harding 2020; McLean et al. 2008).

Table 1 Overview on enzymatic modifications of cyclodipeptides assembled by cyclodipeptide synthases
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Several outstanding reviews have already outlined the variety of cyclodipeptides and their derivatives (Borgman et al. 2019; Canu et al. 2020; Giessen and Marahiel 2015; Moutiez et al. 2017). During the last years, CDPS-associated P450s got more and more attraction. Seventeen members from this enzyme family have been proven to catalyse intriguing reactions. Their catalytic spectrum ranges from hydroxylation of a tertiary carbon, aromatisation, intramolecular C-C bond formation and DKP dimerisation, to transfer of nucleobases to a DKP unit, as exemplarily given in Fig. 2.

Fig. 2
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Examples of modification reactions catalysed by CDPS-associated P450s (highlighted in red)

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Properties of P450s

Cytochrome P450 enzymes represent an enzyme superfamily occurring almost ubiquitously throughout living organisms. In eukaryotes, they are usually bound to membranes, whereas bacterial members appear freely in the cytosol. They contribute a major part in carbon source assimilation, production of secondary metabolites and metabolism of xenobiotics (Chen et al. 2021). P450s have different sizes, cofactors and electron donors as well as various shapes of substrate binding pockets. These features make them versatile enzymes performing a vast variety of stereoselective and stereospecific reactions. The enzymes of this family got their names based on the Soret peak at 450 nm, when the reduced form is complexed with carbon monoxide (Klingenberg 1957). P450s contain a haem acting as a monooxygenase by usually inserting or adding a single oxygen atom onto their substrates. Haem itself is a hexa-coordinated complex consisting of an iron ion in the middle of a planar porphyrin as tetradentate ligand. The iron ion is axially bound to the enzyme on one side and on the opposite side complexed with a molecule water in the resting state. Replacing the water molecule by a reactive oxygen species initiates the P450-catalysed reaction. The central iron ion state is changed from FeIV to FeII (Katagiri et al. 1968).

Bacterial P450s are normally soluble proteins with around 400–500 amino acid residues and can be overproduced in E. coli for in vitro assays. They use usually ferredoxin and ferredoxin reductase as cofactors but are also able to accept electrons from heterologous redox partners (Rudolf et al. 2017).

P450 as hydroxylase in the biosynthesis of bicyclomycin

In the BGC of bicyclomycin from Streptomyces sapporensis, the P450 BcmD acts as a hydroxylase (Fig. 3). The elucidation of the biosynthetic pathway was reported by two different groups in Streptomyces cinnamoneus ATCC 21532 (synonym Streptomyces sapporensis) (Meng et al. 2018; Patteson et al. 2017). A very similar BGC coding for bicyclomycin biosynthetic pathway was found in Pseudomonas aeruginosa SCV20265 (Vior et al. 2018). Several putative bicyclomycin BGCs have also been found in various gram-positive and gram-negative bacteria, suggesting gene transfer events across different bacterial species (Vior et al. 2018).

Fig. 3
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P450 role in the biosynthesis of bicyclomycin in Streptomyces cinnamoneus

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The CDP core of bicyclomycin consisting of l-isoleucine and l-leucine is modified by five non-haem mononuclear iron and 2-ketoglutarate-dependent oxidases and one P450. The bicyclic hemiaminal intermediate is hydroxylated by the P450 BcmD and dehydrogenated by the 2-ketoglutarate-dependent oxidase BcmF (Fig. 3).

P450 as aromatase in the biosynthesis of pulcherriminic acid

Pulcherriminic acid has already been isolated and identified in 1972 from Bacillus subtilis (Uffen and Canale-Parola 1972), whereas its biosynthetic pathway was elucidated 38 years later (Cryle et al. 2010). The BGC of pulcherriminic acid comprises merely two genes coding for a CPDS and a P450 enzyme. The CDP core is assembled from two l-leucine molecules by the CDPS YvmC and altered by the P450 CypX (also termed as CYP134A1) via a three-step oxidative transfer mechanism. The two nitrogen atoms of the DKP ring are oxidised to N-oxides and the DKP ring is aromatised (Fig. 4). This aromatisation is claimed either via hydroxylation and subsequent water elimination or via a direct electron transfer reaction. Afterwards, the oxygen residues undergo a chelation with 2x Fe3+ forming pulcherrimin.

Fig. 4
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P450-catalysed formation of pulcherriminic acid in Bacillus subtilis

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P450 catalysing intramolecular C-C bond formation in the biosynthesis of mycocyclosin

Most sequenced Mycobacterium tuberculosis strains share a two-gene BGC being responsible for the biosynthesis of mycocyclosin, an oxidised cYY product. The CDPS Rv2275 catalyses the condensation of two l-tyrosine molecules to cyclo-(l-Tyr-l-Tyr), which is then converted to mycocyclosin by the P450 enzyme Rv2276, commonly known as CYP121 (Belin et al. 2009). This conversion is an intramolecular C-C bond formation between the ortho-positions to the phenolic residues (Fig. 5). CYP121 shares high sequence homology with P450s catalysing nucleobase transfer reactions (Yu et al. 2018).

Fig. 5
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Conversion of cYY to mycocyclosin by the P450 enzyme CYP121 in Mycobacterium tuberculosis

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P450s as transferases of nucleobases guanine and hypoxanthine

Tryptophan is a frequent target of bacterial tailoring enzymes due to its electron-rich indole moiety (Alkhalaf and Ryan 2015). Very recently, bacterial P450s from several Streptomyces species have been proven to catalyse the linkage of a guanine to a tryptophan residue of CDPs in the biosynthesis of guanitrypmycins and analogues (Liu et al. 2019; Shi et al. 2019; Yu et al. 2018). The elucidation of the biosynthetic pathways was achieved by heterologous expression in Streptomyces coelicolor, precursor feeding experiments and biochemical characterisation with recombinant and purified enzymes.

Four biosynthetic pathways for guanitrypmycins bearing a guanine moieties have been reported so far (Fig. 6), including the two-gene cluster from Streptomyces purpureus NRRL B5737 with the P450 GutD5737 for coupling of guanine with cWW (Yu et al. 2018). Two very similar BGCs consisting of five genes were identified in Streptomyces monomycini NRRL B-24309 and Streptomyces varsoviensis NRRL B-3589. These genes code for four functional enzymes, i.e. CDPS, CDO encoded by two genes, P450 and MT. Both P450 enzymes, GutD24309 from strain B-24309 and GutD3589 from B-3589, catalyse the C3-guaninylation of dehydro CDP derivatives. Subsequent N-methylation at the guanine residue by GutE leads to the formation of cWY derivative guanitrypmycin A2-1. The BGC from strain B-3589 is also responsible for the formation of the cWF derivative guanitrypmycin A1-1 (Liu et al. 2019). An almost identical BGC for guanitrypmycin A2-1, termed purincyclamide in that paper, was later identified in Streptomyces chrestomyceticus NA4264, and the corresponding P450 enzyme was named PcmD (Shi et al. 2019).

Fig. 6
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Coupling reactions of CDPs with nucleobases catalysed by P450s

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Interestingly, the guanine is always attached via C8´ to either N1 or C3 position of the tryptophanyl residue. The first identified GutD5737 catalyses a C-N bond linkage, whereas other three enzymes, GutD24309, GutD3589 and PcmD, a C-C bond formation, followed by a cyclisation between C2 of the indole and N10 of the DKP rings.

In addition to the guanine transfer reactions, the CDPS-associated P450s can also catalyse the coupling of CDP with another nucleobase hypoxanthine. GutD2774 from Streptomyces lavendulae NRRL B-2774 and GutD5414 from Streptomyces xanthophaeus NRRL B-5414 use the same substrate cWW and attach hypoxanthine via its N1´ to C2 of the indole and C2´ to C3, respectively. The main final pathway products are identified correspondingly as guanitrypmycins C3-3 and C3-4 (see Fig. 6). GutD2774 and GutD5414 share sequence identities of 75 and 57% on the amino acid level with GutD5737 and were also found to be capable of using guanine as substrate, resulting in the formation of the minor side products guanitrypmycins C3-1 and C3-2, respectively (Yu et al. 2019). Although guanitrypmycins are unusual bacterial metabolites, no pharmacological and biological properties have been published yet, and their biological function remains therefore unknown. One important reason is their low solubility in aqueous milieu.

P450s as DKP dimerases for C-C bond formation

At least four CDPS-associated P450s catalyse dimerisation of tryptophan-containing CDPs via an intermolecular C-C bond formation between the two tryptophanyl moieties (Fig. 7). Their products are connected via C3 of one tryptophanyl moiety, accompanied by a cyclisation between C2 and N12 based on a Mannich reaction to form a pyrroloindoline system, in analogy to guanitrypmycins mentioned above (Tian et al. 2018).

Fig. 7
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Dimerisation of CDPs catalysed by P450s

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Among the characterised CDP dimerases, NasB from Streptomyces sp. NRRL S-1868 uses cWP and cWA as substrates, resulting in the formation of naseazine A with a C3-C6´ linkage as the mere product (Yu and Li 2019). NascB from Streptomyces sp. CMB-MQ030 (Tian et al. 2018) and NzeB from Streptomyces sp. NRRL F-5053 (Shende et al. 2020) are cWP dimerases for C3-C5´ coupling with naseazine C as the main product. Two two-gene operons from Saccharopolyspora antimicrobica DSM 45119 are responsible for the formation of tetratryptomycins with four tryptophanyl units. TtpB1 catalyses C3-C3´connection between two cWW molecules (Liu et al. 2020). In vitro bioactivity testes revealed that naseazine C showsactivity against chloroquine-sensitive malaria parasites and its derivatives have protective activity against glutamate-induced PC-12 damage (Tian et al. 2018).

P450s as DKP dimerases for C-N bond formation

The aforementioned NzeB also catalyses the N1-C6´dimerisation of two cWP molecules, resulting in the formation of aspergilazine A as minor side product (Shende et al. 2020). Aspergilazine A was identified as the main product of a two-gene BGC from Streptomyces sp. NRRL S-1868, with AspB as the responsible dimerase (Yu and Li 2019).

The second two-gene operon from Saccharopolyspora antimicrobica DSM 45119 is responsible for the formation of tetratryptomycin B. The P450 TtpB2 catalyses dimerisation of cWW via C3-N1´ coupling (see Fig. 7) (Liu et al. 2020). Tetratryptomycin B shows no antibacterial effects on cell lines of E. coli, Bacillus subtilis, Staphylococcus aureus or Pseudomonas aeruginosa.

Structural basis and reaction mechanisms

The first crystal structure of the CDPS-associated P450s CYP121 from Mycobacterium tuberculosis has been solved in 2009 (Belin et al. 2009). Based on the structure together with QM/MM studies, a reaction mechanism with involvement of two radicals was proposed (Dumas et al. 2014). One tyrosyl residue is bound closely to the haem centre in proximity to the key oxidant species of P450s, whereas the other tyrosyl moiety points to the protein surface. The important intermediates bear unpaired electrons at ortho-position to the hydroxyl groups of both phenyl moieties. Intramolecular connection of the two radicals leads to the formation of mycocyclosin (Fig. 5) (Dumas et al. 2014).

Very recently, two groups published the structure of the same P450 from Streptomyces sp. NRRL F-5053, termed NzeB and NasF5053, respectively (Shende et al. 2020; Sun et al. 2020). It was proposed that the dimerisation of cWP catalysed by this enzyme would also be via a radical-mediated mechanism (Fig. 8) (Shende et al. 2020). In contrast to CYP121, this dimerase only forms one radical at nitrogen N1 (Sun et al. 2020) or N12 (Shende et al. 2020) after abstraction of one hydrogen by compound I (intermediate 1). In the mechanism proposed by Shende et al. (2020), the resulting radical then shifts to C3 after cyclisation between N12 and C2 (intermediate 2). The C3 radical attacks subsequently C5’ of the tryptophanyl moiety of the second CDP (intermediate 3), followed by a re-aromatisation via elimination of the C5’-hydrogen mediated by compound II, resulting in naseazine C. Regarding the intermolecular C-N bond formation, a similar radical-mediated mechanism is strongly favoured (Shende et al. 2020). In contrast to the C-C dimerisation, the first hydrogen is abstracted from N1 instead of N12. The subsequent steps take place in analogy to those of C-C bond formation.

Fig. 8
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Proposed mechanism for NzeB reaction, modified from Shende et al. (2020)

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Sequence alignments of NascB, NzeB (synonym NasF5053) and NasS1868 (synonym AspB) revealed four critical amino acid residues in these P450s controlling the regio- and stereoselectivity. Mutation on the key residues at positions 65, 86, 284 and 288 led to the alteration of the regio- and stereospecificity of these enzymes (Sun et al. 2020).

Conclusion

In this review, we summarised the intriguing reactions catalysed by bacterial CDPS-associated P450s like DKP ring aromatisation, CDP dimerisation and nucleobase transfer reactions. These P450s have the advantages that they are soluble proteins and can be easily overproduced in E. coli. Some of them show a flexible substrate tolerance towards CDP analogues (Tian et al. 2018), which could be used for production of designed CDP derivatives by chemoenzymatic and synthetic biological approaches, e.g. by recombination of different genes for CDPS, P450s or for other enzymes like prenyltransferases (Dubois et al. 2019).

Genome sequencing revealed the presence of a large number of cdps-containing BGCs including those with P450s as tailoring enzymes. More than 700 of such BGCs were identified in 93107 prokaryotic genomes (Skinnider et al. 2018). The number of the clusters of interest will undoubtedly increase with the number of ongoing sequencing projects. Targeted gene activation might not only uncover novel natural products but also reveal new functions of tailoring enzymes including P450s.