Streptomyces spp. are known to produce various types of biologically active compounds including antibiotics, antiparasitic agents, herbicides and immunosuppressants. P450 (cytochrome P450) enzymes may have key roles in these biosynthetic and biotransformation reactions. Recent genomic analysis of Streptomyces coelicolor A3(2) indicates that S. coelicolor may have six ferredoxins (Fdxs), four putative Fdx reductases (FdRs) and 18 P450 genes. However, there are few clues to explain the mechanisms and functions of Streptomyces P450 systems. To solve these questions, we have expressed and purified five S. coelicolor P450s, four FdRs and six Fdxs in Escherichia coli. Of the purified P450s, CYP105D5 has fatty acid hydroxylation activity in a system reconstituted with putidaredoxin reductase and Fdx4 or with spinach FdR and spinach Fdx, although the reconstitutions with FdR2 or FdR3 and any of the Fdxs did not support CYP105D5-catalysed oleic acid hydroxylation. Elucidation of the detailed mechanisms of electron transport system for Streptomyces P450 may provide the perspective for usefulness of P450s as a biocatalyst.

Introduction

Streptomyces is a genus of Gram-positive bacteria that grows in soil, marshes and coastal marine habitats. Streptomyces grows as a mycelium of branching hyphal filaments, like fungi, and reproduce in a mould-like manner by sending up aerial branches that turn into spore chains. Morphological differentiation in Streptomyces requires the specialized co-ordination of metabolism, resulting in production of an array of complex secondary metabolites [1].

The most important property of Streptomyces is its ability to produce various antibiotics and bioactive compounds valuable for medical, veterinary and agricultural use. Streptomyces spp. produce more than 50% of known antibiotics and they also produce numerous antitumour agents, antifungal agents, antiparasitics and immunosuppressants, as well as antibiotics (Table 1). Streptomyces also catalyses oxidative transformation of chemicals such as alkaloids, coumarins, retinoids and other complex xenobiotics. P450 (cytochrome P450) enzymes may have key roles in these biosynthetic and biotransformation reactions.

Table 1
Well-known bioactive chemicals produced in Streptomyces

FK506 is an immunosuppressant macrolide.

ChemicalsStreptomyces speciesFunction
Adriamycin S. peucetius Antitumour agent 
Amphotericin B S. nodosus Antifungal agent 
Avermectin S. avermitilis Antiparasitic agent 
Clavulanic acid S. clavuligerus β-Lactamase inhibitor 
Erythromycin S. erythreus Antibacterial agent 
FK506 S. tsukubaensis Immunosuppressant 
Neomycin S. fradiae Antibacterial agent 
Pravastatin S. carbophilus Anticholesterol agent 
Rapamycin S. hygroscopicus Immunosuppressant 
Rifamycin S. mediterranei Antibacterial agent 
Streptomycin S. griseus Antibacterial agent 
Tetracyclin S. rimosus Antibacterial agent 
Vancomycin S. orientalis Antibacterial agent 
ChemicalsStreptomyces speciesFunction
Adriamycin S. peucetius Antitumour agent 
Amphotericin B S. nodosus Antifungal agent 
Avermectin S. avermitilis Antiparasitic agent 
Clavulanic acid S. clavuligerus β-Lactamase inhibitor 
Erythromycin S. erythreus Antibacterial agent 
FK506 S. tsukubaensis Immunosuppressant 
Neomycin S. fradiae Antibacterial agent 
Pravastatin S. carbophilus Anticholesterol agent 
Rapamycin S. hygroscopicus Immunosuppressant 
Rifamycin S. mediterranei Antibacterial agent 
Streptomycin S. griseus Antibacterial agent 
Tetracyclin S. rimosus Antibacterial agent 
Vancomycin S. orientalis Antibacterial agent 

The genome project of the Streptomyces coelicolor A3(2) has provided valuable genetic information about components of Streptomyces P450 electron transport systems. This review exploits some of the recent findings about the mechanisms of P450 electron transport systems in S. coelicolor.

Streptomyces P450s

P450s are a superfamily of haem-containing mono-oxygenases involved in the oxidative metabolism of a wide range of endogenous and xenobiotic chemicals. P450s also play important roles in the biosynthesis of antibiotics and other biologically active molecules in bacteria, fungi and plants as well as in the biosynthesis of steroids, fatty acids, prostaglandins and other physiologically important chemicals in animals [2]. All known P450s are multicomponent enzymes containing a haem component with associated reductase components. Electrons are delivered from the reduced pyridine nucleotide coenzymes NAD(P)H to the P450 via a FAD-containing FdR [Fdx (ferredoxin) reductase] and an iron–sulfur protein Fdx in the most bacterial and mitochondrial P450s (class I). The class II P450 systems such as eukaryotic microsomal P450s have membrane-bound NADPH-dependent P450 reductase containing FAD and FMN. Electrons from NADPH are transferred to the FAD of P450 reductase and on to the P450 via the FMN of P450 reductase. However, bacterial P450s such as CYP102A1, CYP102A2 and CYP102A3 from Bacillus species (class III) contain NADPH-dependent diflavin reductase (FAD and FMN) and P450 in one continuous polypeptide. Class IV P450s, recently discovered in Rhodococcus, are one-component enzymes comprising an NADPH-dependent FMN-containing FdR and Fdx fused to the haem domain [2a].

In common with most other bacterial P450s, those from Streptomyces species are of the classical class I system made up of a FAD-containing FdR, Fdx and the P450. Streptomyces P450s are involved in polyketide biosynthesis by catalysing the stereo- and regio-specific oxidative modification of macrolide antibiotics such as erythromycin, tylosine and oleandomycin [3]. Because antibiotic potency can be significantly enhanced by P450-mediated reaction, the biotechnological value of these P450s has been recognized. P450s from Streptomyces strains have also been shown to be necessary for synthesis of the antifungal agents, the antitumour agents, the antiparasitic agents and the immunosuppressants as well as the antibacterial agents [3a].

Genome sequencing projects of S. coelicolor A3(2) revealed 18 putative P450 genes among 7825 genes in the linear 8.7 Mb chromosome (CYP51, CYP102B1, CYP105D5, CYP105N1, CYP107P1, CYP107T1, CYP107U1, CYP154A1, CYP154C1, CYP155A1, CYP156A1, CYP156B1, CYP157A1, CYP157B1, CYP157C1, CYP158A1, CYP158A2 and CYP159A1) [4,5]. However, the endogenous functions and biological roles of each P450 remain largely unknown. Heterologous expression of these P450 genes in Escherichia coli shows that all expressed P450s are soluble, locating to the cytosol. The expression levels in E. coli were quite different among P450s. Expression levels of CYP51, CYP102B1, CYP105N1, CYP157B1 and CYP157C1 were very low (<10 nmol/l of culture), while P450s such as CYP105D5, CYP154A1 and CYP154C1 showed very high level of expression (∼6500 nmol/l of culture). Among them, CYP105D5, CYP107U1, CYP154A1, CYP154C1 and CYP158A2 were purified (Figure 1) and the crystal structures of CYP154C1, CYP154A1 and CYP158A2 have been determined [68].

Spectra of purified Streptomyces CYP105D5 from E. coli

Figure 1
Spectra of purified Streptomyces CYP105D5 from E. coli

(A) Absolute spectrum. (B) Fe2+-CO versus Fe2+ difference spectrum.

Figure 1
Spectra of purified Streptomyces CYP105D5 from E. coli

(A) Absolute spectrum. (B) Fe2+-CO versus Fe2+ difference spectrum.

CYP154C1 shows in vitro catalytic activity towards macrolide intermediates such as YC-17 and narbomycin, which are converted into methylmycin, neomethylmycin, novamethylmycin and pikromycin by pikC in Streptomyces venezuelae. However, the biological function of CYP154C1 in S. coelicolor is still unknown because cells of S. coelicolor do not produce these antibiotics [6]. In contrast, CYP154A1 shows weak binding towards narbomycin, with a Kd of over 1 mM, and no catalytic activity towards narbomycin is observed. CYP158A2 can produce dimeric and trimeric products from the substrate flaviolin and two flaviolin molecules occupy the active site in the substrate-bound structure of CYP158A2 [8].

A putative function of CYP105D5 in xenobiotic metabolism has been suggested because CYP105D5 shows a high level of similarity to CYP105D1 in Streptomyces griseus. CYP105D1 is able to oxidize a diverse array of xenobiotics such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics [9]. To determine the catalytic activity of CYP105D5, binding of CYP105D5 with fatty acids such as arachidonic, lauric, linoleic, myristic, oleic and palmitic acids was studied. These fatty acids could interact with CYP105D5 with Kd values of less than 1 μM. Hydroxylation of oleic, palmitic and lauric acids by CYP105D5 was also suggested. Recently, hydroxylation of testosterone at the 2β, 15β and 17 positions using E. coli cells expressing CYP105D5 has been reported [10]. Thus a role of CYP105D5 in xenobiotic metabolism should be considered.

Streptomyces Fdxs and FdRs

Fdxs are small, acidic soluble iron–sulfur proteins that are widely distributed in bacteria, plants and animals. Most Fdxs having [Fe-S] clusters as their redox-active site play a key role in electron transfer or in metabolic reactions, because the ability to delocalize electron density over both Fe and S atoms makes [Fe-S] clusters suitable for mediating electron transport [11]. Fdxs can be subdivided into several different classes according to the presence of specific types of [Fe-S] clusters. In bacteria, adrenodoxin-type Fdxs such as putidaredoxin, terpredoxin and rhodocoxin contain [2Fe-2S] iron–sulfur clusters as a redox-active group. The prosthetic group of these Fdxs consists of two iron atoms each ligated by thiol groups of two cysteine residues and bridged by two sulfide ions. These bacterial Fdxs are involved in the transfer of electrons from NADH to P450 enzymes by means of FdR. In contrast, bacterial-type Fdxs have an [3Fe-4S] or [4Fe-4S] iron–sulfur cluster as their redox centres. These proteins are widely distributed in bacteria and archaea and also act as electron carriers. Of these proteins, two [3Fe-4S] Fdxs have been isolated and spectroscopically characterized from Streptomyces griseolus. Fdx containing [3Fe-4S] cluster from Streptomyces clavuligerus was shown to be involved in clavulanic acid biosynthesis. Mycobacterium tuberculosis [3Fe-4S] Fdx can pass electrons to CYP51, a 14α-sterol demethylase.

FdRs are ubiquitous monomeric enzymes having one molecule of non-covalently bound FAD as prosthetic groups. In addition to the FAD-binding domain, all of the FdRs have the NAD(P)H-binding domain [12]. They catalyse the reversible electron transfer between NAD(P)H and the Fdx or flavodoxin. Only a few FdRs of bacterial P450 systems have been purified and characterized because of their unstable nature and the relatively low level of expression. Putidaredoxin reductase is a component of the soluble P450cam system from Pseudomonas putida. The protein can function as an NADH-dependent one-electron carrier from NADH to [2Fe-2S] cluster-containing putidaredoxin, which, in turn, transfers electrons to the terminal oxygenase P450cam. Recently, the crystal structure of putidaredoxin reductase was solved and the structural basis for putidaredoxin reductase and putidaredoxin electron transfer complex has been studied [13]. NADH-dependent FdR has been purified from S. griseus cells grown in soya-bean flour-enriched medium, and cytochrome c reduction by the FdR is enhanced by the addition of S. griseus Fdx [14]. FdR purified from Mycobacterium sp. strain HE5 also has been characterized as a NADH-dependent, FAD-containing protein. However, FdR of M. tuberculosis is an NADPH-dependent FAD-containing protein.

Using genomic information about S. coelicolor A3(2), six Fdxs and four FdR genes were cloned and expressed in E. coli [5,15]. Of the six purified Fdxs, Fdx4 may be one of the candidates for natural electron transfer partner protein for Streptomyces P450s because it accepts electrons from putidaredoxin reductase or spinach FdR and transfers to CYP105D5. Fdx4 is shown to directly bind to CYP105D5. Reconstitution with putidaredoxin reductase or spinach FdR and Fdx4 supports CYP105D5-mediated oleic acid hydroxylation. Conformational changes of Fdx4 and CYP105D5 upon formation of a complex structure and essential amino acid residues for the conformational changes need to be determined.

Although FdRs from S. coelicolor have been purified, the detailed structural and enzymatic properties are not completely determined yet. Co-expression of S. griseolus CYP105A1 and 105B1 and their Fdxs, Fd1 and Fd2, and S. coelicolor FdR1 and FdR2 in E. coli enhances correctly folded P450s and dealkylation of 7-ethoxycoumarin [16]. Streptomyces lividans TK24 cells expressing S. griseolus CYP105D1, Fdx and S. coelicolor FdR1 also show 7-ethoxycoumarin O-dealkylation activity in vivo. However, purified FdR2 and FdR3 show a relatively lower cytochrome c reduction activity at neutral pH range and did not support CYP105-catalysed fatty acid hydroxylation with any isolated Fdx. The possibilities of newly expressed FdR1 and FdR4 as a natural electron donor protein for CYP105D5 should be addressed.

Future perspectives

There are considerable challenges to use P450s for the catalysis of biotransformation. Especially, in Streptomyces, P450s are involved in biosynthesis of various secondary metabolites including antibiotics, antitumour agents and other clinically valuable chemicals. To evaluate P450s as a tool for biocatalysis, the understanding of the mechanisms of the P450 system will be required. Although recent progress in studying P450 structures, protein–protein interactions and specialized activities of enzymes provides new perspectives with respect to elucidating the functions of P450s in Streptomyces species, many questions still remain. Clearly, we are only beginning to learn about the Streptomyces P450 systems. Future studies should be focused on determining the natural components of P450 systems and their interactions in S. coelicolor.

8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology: Independent Meeting held at Swansea Medical School, Swansea, Wales, U.K., 23–27 July 2006. Organized and Edited by D. Kelly, D. Lamb and S. Kelly (Swansea, U.K.).

Abbreviations

     
  • Fdx

    ferredoxin

  •  
  • FdR

    Fdx reductase

  •  
  • P450

    cytochrome P450

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