Phenoxazinone synthase: what's in a name
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Phenoxazinone synthase: what’s in a name? Marilize Le Roes-Hill, Candice Goodwin and Stephanie Burton Biocatalysis and Technical Biology Group, Department of Chemical Engineering, University of Cape Town, Private Bag 1, Rondebosch, 7701, Cape Town, South Africa The name phenoxazinone synthase (PHS, 2-aminophe- nol:oxygen oxidoreductase, EC 1.10.3.4) is used for the enzyme catalysing the oxidative coupling of substituted o-aminophenols to produce phenoxazinones. This review reveals that the traditional classification of PHS conflicts with recent sequence-based information that shows its relationship with two distinct copper protein groups. Different PHS roles, namely spore pigmentation in Streptomyces antibioticus (phsA) and biosynthesis of the antibiotic grixazone in Streptomyces griseus subsp. griseus (GriF), indicate an example of convergent evol- ution. Here, we review the classification, distribution and roles of PHSs, comparing them with copper oxidases at genetic and structural levels and exploring their potential application in the production of new antibiotics. In search of a phenoxazinone synthase Traditionally in the field of biochemistry, enzymes were classified according to their biochemical activity, based on the characterization of the substrates converted and the products generated. Thus, enzymes shown to catalyse closely related reactions were assumed to be closely related structurally. In recent years, as sequence-based approaches have shed light on the genetic relationships between proteins, new patterns have become apparent where structure and function might have evolved conver- gently from distant routes [1,2]. Conversely, information arising from modern protein structure elucidation technol- ogies allows us to identify structural relationships between proteins with diverse biochemical function. Phenoxazinone synthase (PHS) has been known for several decades; it was first recognized for its proposed role in antibiotic production and is associated with the biosynthesis of phenoxazinone core metabolites (Box 1). Its history began in the 1960 s when the discovery of new antibiotics was recognized as a pressing need, but researchers did not yet have the advantages of present day molecular and crystallographic technologies to assist in understanding and exploiting microbial production of secondary metabolites. More recent research has shown us that PHS is something of an enigma, presenting new questions as to the role, nature and class of a group of enzymes all called PHS [3,4]. Through a survey of the historical development of information on the PHS enzymes, together with a correlation to recent information, this review provides a view of the PHS enzymes, their roles, relationships and potential applications. PHS as a member of EC 1.10.3.4 PHS is represented in the traditional Enzyme Commission classification systems in the class EC 1.10.3.4 (2-amino- phenol:oxygen oxidoreductase). The comprehensive electronic enzyme information system, BRENDA (http:// www.brenda-enzymes.info/index.php4), records several synonyms for PHS (isophenoxazine synthase, esophenox- azine synthase, o-aminophenol oxidase, GriF, o-aminophe- nol:O 2 oxidoreductase and oxidase, o-aminophenol), but varying roles of the enzyme have been reported, from antibiotic production (GriF) and the biosynthesis of cate- chols (isophenoxazine synthase) to the production of fungal metabolites (esophenoxazine synthase). In comparing reports of PHS enzymes, anomalies emerge, particularly on consideration of recent molecular and structure–func- tion information [3,5]. The first PHS reported, phsA, from Streptomyces antibioticus [6], is now known to differ mark- edly from the PHS enzyme called GriF, from Streptomyces griseus subsp. griseus [3]. Furthermore, GriF is classified as a member of EC 1.10.3.4 in several databases, but the S. antibioticus phsA has not been included in most of these. The phenoxazinone synthase reaction It is generally agreed that PHSs catalyse the oxidative condensation of o-aminophenols, but it might also oxidize o-diphenols and p-aminophenols, although possibly at a slower rate [3,7]. A mechanism for the reaction catalysed by phsA in S. antibioticus has been proposed, as shown in Figure 1a: the o-aminophenol is first oxidized by two electrons to the quinone imine, which then conjugates to a second o-aminophenol molecule while still bound to the enzyme. This product is further oxidized by two electrons to give rise to the p-quinone imine. The last two steps of the reaction, another conjugation to generate the tricyclic structure and a final two-electron oxidation to yield the 2-aminophenoxazinone product, are thought to be non- enzymatic [8]. The mechanism of this complex six-electron oxidation was determined by using a variety of substituted o-aminophenols designed to block the reaction at inter- mediate stages [9]. The reaction catalysed by GriF in S. griseus subsp. griseus is slightly different. The enzyme catalyses the oxidation of an o-aminophenol, 3,4-aminohy- droxybenzaldehyde, to form the corresponding quinone imine. This compound is then non-enzymatically conju- gated by the –SH group of N-acetylcysteine, oxidized by Review E-mail addresses: [email protected], [email protected] 248 0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.01.001 Available online 4 March 2009
Transcript of Phenoxazinone synthase: what's in a name
Phenoxazinone synthase: what’s in aname?Marilize Le Roes-Hill, Candice Goodwin and Stephanie Burton
Biocatalysis and Technical Biology Group, Department of Chemical Engineering, University of Cape Town, Private Bag 1,
Rondebosch, 7701, Cape Town, South Africa
Review
The name phenoxazinone synthase (PHS, 2-aminophe-nol:oxygen oxidoreductase, EC 1.10.3.4) is used for theenzyme catalysing the oxidative coupling of substitutedo-aminophenols to produce phenoxazinones. Thisreview reveals that the traditional classification of PHSconflicts with recent sequence-based information thatshows its relationship with two distinct copper proteingroups. Different PHS roles, namely spore pigmentationin Streptomyces antibioticus (phsA) and biosynthesis ofthe antibiotic grixazone in Streptomyces griseus subsp.griseus (GriF), indicate an example of convergent evol-ution. Here, we review the classification, distributionand roles of PHSs, comparing them with copperoxidases at genetic and structural levels and exploringtheir potential application in the production of newantibiotics.
In search of a phenoxazinone synthaseTraditionally in the field of biochemistry, enzymes wereclassified according to their biochemical activity, based onthe characterization of the substrates converted and theproducts generated. Thus, enzymes shown to catalyseclosely related reactions were assumed to be closely relatedstructurally. In recent years, as sequence-basedapproaches have shed light on the genetic relationshipsbetween proteins, new patterns have become apparentwhere structure and function might have evolved conver-gently from distant routes [1,2]. Conversely, informationarising frommodern protein structure elucidation technol-ogies allows us to identify structural relationships betweenproteins with diverse biochemical function.
Phenoxazinone synthase (PHS) has been known forseveral decades; it was first recognized for its proposedrole in antibiotic production and is associated with thebiosynthesis of phenoxazinone core metabolites (Box 1). Itshistory began in the 1960 s when the discovery of newantibiotics was recognized as a pressing need, butresearchers did not yet have the advantages of presentday molecular and crystallographic technologies to assistin understanding and exploiting microbial production ofsecondary metabolites. More recent research has shown usthat PHS is something of an enigma, presenting newquestions as to the role, nature and class of a group ofenzymes all called PHS [3,4]. Through a survey of thehistorical development of information on the PHSenzymes, together with a correlation to recent information,
E-mail addresses: [email protected], [email protected]
248 0167-7799/$ – see front matter � 2009 Elsevier
this review provides a view of the PHS enzymes, theirroles, relationships and potential applications.
PHS as a member of EC 1.10.3.4PHS is represented in the traditional Enzyme Commissionclassification systems in the class EC 1.10.3.4 (2-amino-phenol:oxygen oxidoreductase). The comprehensiveelectronic enzyme information system, BRENDA (http://www.brenda-enzymes.info/index.php4), records severalsynonyms for PHS (isophenoxazine synthase, esophenox-azine synthase, o-aminophenol oxidase, GriF, o-aminophe-nol:O2 oxidoreductase and oxidase, o-aminophenol), butvarying roles of the enzyme have been reported, fromantibiotic production (GriF) and the biosynthesis of cate-chols (isophenoxazine synthase) to the production of fungalmetabolites (esophenoxazine synthase). In comparingreports of PHS enzymes, anomalies emerge, particularlyon consideration of recent molecular and structure–func-tion information [3,5]. The first PHS reported, phsA, fromStreptomyces antibioticus [6], is now known to differ mark-edly from the PHS enzyme called GriF, from Streptomycesgriseus subsp. griseus [3]. Furthermore, GriF is classifiedas a member of EC 1.10.3.4 in several databases, but the S.antibioticus phsA has not been included in most of these.
The phenoxazinone synthase reactionIt is generally agreed that PHSs catalyse the oxidativecondensation of o-aminophenols, but it might also oxidizeo-diphenols and p-aminophenols, although possibly at aslower rate [3,7]. A mechanism for the reaction catalysedby phsA in S. antibioticus has been proposed, as shown inFigure 1a: the o-aminophenol is first oxidized by twoelectrons to the quinone imine, which then conjugates toa second o-aminophenol molecule while still bound to theenzyme. This product is further oxidized by two electronsto give rise to the p-quinone imine. The last two steps of thereaction, another conjugation to generate the tricyclicstructure and a final two-electron oxidation to yield the2-aminophenoxazinone product, are thought to be non-enzymatic [8]. The mechanism of this complex six-electronoxidation was determined by using a variety of substitutedo-aminophenols designed to block the reaction at inter-mediate stages [9]. The reaction catalysed by GriF in S.griseus subsp. griseus is slightly different. The enzymecatalyses the oxidation of an o-aminophenol, 3,4-aminohy-droxybenzaldehyde, to form the corresponding quinoneimine. This compound is then non-enzymatically conju-gated by the –SH group of N-acetylcysteine, oxidized by
Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.01.001 Available online 4 March 2009
Box 1. The phenoxazinone core structure
The phenoxazinone core (Figure Ia) has been found in various
biological systems including pigments produced by diverse organ-
isms such as insects, fungi and Australian marsupials. It seems to
participate in a mechanism to protect mammalian tissue from
oxidative damage [70] and forms the core structure of certain
antibiotics (see ‘Phenoxazinone antibiotics’ in the main text). It is
also considered to contribute to the activity of phenoxazinone
antibiotics, allowing the compounds to intercalate nucleic acids,
which is why so many phenoxazinones are effective anticancer
agents [65]. The core structure is also found in the dyes resazurin
and resorufin [73], and some phenoxazinone compounds are used
as substrate probes for the detection of enzymes [74] and the
amplification of the antibiotic activity of other antibiotics [75].
Phenoxazinones have also been detected as by-products in the
growth medium of Pseudomonas putida strain TW3, when the strain
is growing on 4-nitro-substituted substrates [76]. It is therefore not
surprising that various enzymes have been reported to be involved
in the production of the core structure (see main text).
One example from nature is 3-hydroxyanthranilic acid (3-HAA)
(Figure Ib), which is the precursor of the laccase-catalysed formation
of the phenoxazinone derivative cinnabarinic acid (CA) in the fungus
Pycnoporus cinnabarinus [10,70]. Members of this genus are
remarkable laccase producers, and the three pigments isolated
from this genus (cinnabarinic acid, cinnabarin, and tramesanguin)
differ only in the oxidation state of their substituents. This variation
might be due to oxidative coupling of different o-aminophenol
precursors, with the substituents having either an alcohol, aldehyde
or carboxyl group. Furthermore, the production of these precursors
can be strain specific or culture condition dependent [70].
Figure I. Phenoxazinones. (a) Phenoxazinone core structure. (b) Synthesis of
phenoxazinone derivatives from 3-hydroxyanthranilic acid (3-HAA).
Figure 1. Phenoxazinone synthase reaction schemes. (a) The reaction catalysed by
phsA from Streptomyces antibioticus is a six-electron oxidation of o-aminophenol
to phenoxazinone with the concomitant reduction of oxygen to water. The reaction
involves two distinct reactions: (i) two 2-aminophenol + dioxygen ! 2 6-
iminocyclohexa-2,4-dienone + two water (enzymatic); and (ii) two 6-
iminocyclohexa-2,4-dienone + oxidant ! 2-aminophenoxazin-3-one + reduced
oxidant (non-enzymatic) [8]. (b) The reaction catalysed by GriF from
Streptomyces griseus subsp. griseus involves the oxidation of 3-amino-4-
hydroxybenzaldehyde to its corresponding quinone imine followed by the non-
enzymatic conjugation of the –SH group of N-acetylcysteine. The resultant
compound is oxidized to its quinone imine enzymatically and is then dimerized
non-enzymatically with another quinone imine [3]. The areas highlighted with
different colours indicate the changes made to the molecules during the oxidation
reactions. Where two different colours are used in the same molecule, it is to
indicate the different parts of the molecule affected by the reaction. Molecules
introduced into the reactions have also been highlighted.
Review Trends in Biotechnology Vol.27 No.4
GriF to a quinone imine and dimerized non-enzymaticallywith another quinone imine molecule [3] (Figure 1b).
Interestingly, the reactions catalysed by phsA aresimilar to those catalysed by a variety of other proteins,including the enzymes laccase [10], tyrosinase [11], per-oxidase [12], cytochrome c and cytochrome oxidase [13] andcerruloplasmin [11], all of which have been reported tocatalyse the oxidation of the o-aminophenol substrate, 3-hydroxyanthranilic acid (3-HAA). Human haemoglobinhas been shown to be involved in 3-HAA metabolismand in the formation of phenoxazinones within humanerythrocytes [14]. Furthermore, the non-enzymatic for-mation of phenoxazinones has been exploited in the designof functional models to predict the mechanism of 2-ami-nophenol oxidation by enzymes [15,16].
Discovery of PHS and its distribution in naturePHSs are not widely distributed in nature and have onlybeen described from a few sources. PhsA was firstdescribed in 1961 byWeissbach and Katz [6], who detectedthe enzyme in a well-known antibiotic-producing strain ofS. antibioticus. It was believed that phsA formed an essen-tial part in the synthesis of the chromopeptide antibioticactinomycin through the production of the chromophoreactinocin, but this was later disproved [4]. However, theinterest in actinomycin continues owing to its use as ananticancer agent: today, Actinomycin D (generic names:Dactinomycin, Actinomycin IV or Actinomycin C1; tradename: Cosmegen1) is commonly used in the treatment of awide range of cancers (http://www.chemocare.com/bio/actinomycind.asp).
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In 2006, Suzuki et al. [3] reported the discovery ofanother PHS responsible for the formation of the chromo-phore of grixazone, a parasiticide compound produced byS.griseus subsp. griseus (see section on Phenoxazinone anti-biotics). Suzuki et al. [3] cloned the grixazone biosyntheticgene cluster and identified two key gene products, GriEand GriF, which are involved in the formation of thephenoxazinone core structure. From mutation and bioca-talytic studies, they concluded that GriF is an o-amino-phenol oxidase and is a member of EC 1.10.3.4.
Since the early reports of a PHS in streptomycetestrains, the enzyme has also been reported in two plantspecies, Tecoma stans (the yellow trumpetbush) [17] andBauhinia monandra (the dwarf orchid tree referred to as‘Napoleon’s plume’), where PHS was reported to beinvolved in catechol production [18], as well as in thewood-rotting bracket fungus Pycnoporus coccineus [19].Further, a PHS was also implicated in the production ofxanthommatin, the brown eye pigment of Drosophila mel-anogaster, and in the production of ommochromes in thebuckeye butterfly, Precis coenia [20]. However, a study oneye pigmentation in yellow fever mosquito pupae (Aedesaegypti) showed that it is unlikely that this enzyme isinvolved in pigmentation, and the involvement of a PHSin pigment production in insects therefore still needs to beverified [20].
The name ‘cinnabarinate synthase’ was once consideredto be a synonym for PHS: both enzymes utilize 3-HAA as asubstrate with the subsequent reduction of oxygen towaterand the production of cinnabarinic acid (CA; 2-amino-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid). CA hasattracted much interest because there might potentiallybe a link between its occurrence and bladder tumourformation in humans [21]. Cinnabarinate synthase hasbeen detected in nuclear fractions of rat liver [22] andbaboon liver [21] and in the soluble liver fractions ofpoikilothermic invertebrates and poikilothermic animals[23,24]. However, the work of Savage and Prinz [21]showed that the enzyme has the same function as a cat-alase and therefore cinnabarinate synthase was reclassi-fied to the catalase superfamily (EC 1.11.1.6); these resultswere subsequently confirmed in studies on silkworms [25].
Thus, it seems that there are only a few enzymes thatcan be considered to be true PHS enzymes. Furthermore,from the information available, the four PHSs listed inenzyme databases (the PHSs from B. monandra, T. stans,P. coccineus and S. griseus subsp. griseus) and the phsAfrom S. antibioticus differ from each other in many funda-mental aspects, including substrate selectivity, cofactordependence, inhibition profiles and molecular structure(Table 1). In particular, reports [3,5] have shown that onlythe bacterial PHS enzymes are copper-containing proteins,making them part of the large group of copper oxidaseenzymes that catalyse oxidation–reduction reactions, inwhich Cu2+ plays a key part in electron transfer and theresultant reduction of oxygen to water [8]. In contrast tothis, the PHS from the plant Tecoma stans is activated byMn2+ and requires flavin adenine dinucleotide (FAD) formaximal activity [17]; the PHS from P. coccineus requiresMn2+ and riboflavin 50-monophosphate and is typicallyinactive in the absence of flavin mononucleotide (FMN)
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[19]; and the PHS of B. monandra requires no co-factor[18]. Although such discrepancies can often be resolvedthrough the comparison of gene sequences that encode forthe proteins, the lack of genetic information on the plantand fungal PHSs precludes such a comparison. Theremainder of this review focuses on the well-characterizedcopper-containing bacterial PHSs from S. antibioticus(phsA) and S. griseus subsp. griseus (GriF).
PhsA and GriF as copper oxidasesCopper is considered to be one of the essential traceelements found in nature, and it is speculated that theoxidation of Cu(I) to Cu(II) in the early stages of biologicaldevelopment contributed to the evolution of copper-con-taining proteins with the ability to catalyse oxidation–
reduction reactions [8,26]. This large group of enzymesincludes monooxygenases, dioxygenases and oxidases, allof which have important roles in various biological pro-cesses, such as melanin and antibiotic production, amongothers [26]. Based on the electronic structure and geometryof the enzyme active site, as well as the visible, UV andelectron paramagnetic resonance spectra, copper siteshave been grouped into three classes named as: type 1(blue), type 2 (normal) and type 3 (coupled binuclear)[8,26,27]. Enzymes containing copper sites are sub-sequently classified according to the type of copper sitespresent in their active site: enzymes with mononucleartype 2 copper ions (e.g. amine oxidase and galactoseoxidase); enzymes with binuclear type 3 copper ions (e.g.catechol oxidase and tyrosinase); andmulticopper oxidases(MCOs; e.g. laccase), which might contain type 1, type 2and type 3 copper ions [27].
PhsA from S. antibioticus as a multicopper oxidase
The MCO family consists of a group of enzymes that aredefined by sequence homology, spectroscopy and reactivity.This large group of enzymes includes laccase (EC 1.10.3.2),ascorbate oxidase (EC 1.10.3.3), ceruloplasmin (EC1.16.3.1), bilirubin oxidase (EC 1.3.3.5), dihydrogeodinoxidase, sulochrin oxidase (EC 2.10.3.8) and the ferroustransporter protein FET3 [8,28].
During their study on a laccase from Streptomyceslavendulae REN-7, Suzuki et al. [29] showed that theamino acid sequence of this laccase had a 76% identityto the phsA of S. antibioticus. This laccase is thought tohave a role in either spore pigmentation or melanin pro-duction, similar to the role proposed for the phsA in S.antibioticus [4]. A protein BLAST [30] analysis of phsAconfirmed it had greatest sequence similarity to the aminoacid sequence of the S. lavendulae laccase, followed by thelaccase sequences of Streptomyces cyaneus and Strepto-myces clavuligerus. This discovery prompted researchersto classify phsA as a potential member of the MCO family.Further analysis of the protein has confirmed that phsAfrom S. antibioticus is a blue MCO [5,26,31].
Members of the MCO family belong to the blue copperprotein group and have between one and six copper atomsper molecule; they typically catalyse the oxidation oftheir substrates, with the concomitant reduction of mol-ecular oxygen to water [28]. MCO family members arewidespread in nature [8]. The electron transfer during
Table 1. Enzymes classified as PHS (EC 1.10.3.4)a
Source
Plant Fungal Bacterial
Bauhinia monandra Tecoma stans Pycnoporus
coccineus
Streptomyces griseus
subsp. griseus (GriF)
Streptomyces antibioticus
(phsA)
Substrates 2-aminophenol + O2 2-aminophenol
+ O2
2-aminophenol
+ O2
2-amino-4-methylphenol
+ O2
3-hydroxyanthranilic
acid + O2
3,4-dihydroxybenzaldehyde
+ O2
4-methyl-3-hydroxyanthranilic
acid + O2
3-amino-4-
hydroxybenzaldehyde + O2
3-hydroxykynurenine + O2
Catechol + O2
4-methyl-3-hydroanthranilic
acid methyl ester + O2
L-DOPA + O2 O-aminophenol + O2
o-aminophenol + O2 L-DOPA + O2
protocatchuic acid + O2 Catchol + O2
Ferrocyanide + O2
Thiophenol + O2
Role Catechol production Unknown Pigment
production
Grixazone production Spore coat pigmentation?
Melanin production?
Co-factor
required
None FAD and Mn2+ Mn2+ and
riboflavin-50-
phosphate
Cu2+ Cu2+
Inhibitors Metal ions: Ag+, Cu2+,
Fe2+, Hg2+
2,3-dimercaptopropanol
3-hydroxyanthranilic acid
Anthranilic acid
Ascorbic acid
Atebrin
Azide
Cyanide
Cysteine
Glutathione
N-ethylmaleimide
p-hydroxymercuribenzoate
Metal ions: Co2+,
Fe3+, Hg2+
Mg2+
Atebrin
Cyanide
Ascorbic acid
CuSO4
Cysteine
FeSO4
Glutathione
N-ethylmaleimide
Na3AsO3
NaBH4
o-aminophenol
(>0.6 mM)
p-chloro-
mercuribenzoate
3-amino-4-
hydroxybenzensulfonic
acid
4-hydroxy-3-
nitrobenzaldehyde
Aniline
L-tyrosine
O-nitrophenol
P-hydroxybenzaldehyde
Phenol
Metal ions: Zn, Fe2+, Cu2+
K
NH4
Azide
Cyanide
EDTA
8-hydroxyquinoline
Sodium
diethyldithiocarbamate
a,a0-dipyridyl
Gene expression is subject
to glucose repression
pH optimum 6.2 6.2 ND 8.5–10.5 5.0–5.2
Temperature
optimum
40 8C 45 8C ND 55 8C 42 8C
Amino acid
sequence
homology
ND ND ND Tyrosinase
= type 3 multicopper
oxidase
Laccase
CotA
Bilirubin oxidase
= blue multicopper oxidase
Number of
copper atoms
associated
with structure
No Cu No Cu No Cu Two type 3 Five: one type 1 (blue), two
type 2 and one binuclear type
3; the fifth copper atom is a
type 2 and has a structural
role
Refs [18] [17] [19] [3] [7,28,31,72]
Abbreviation: ND, not determined.aFor further information, see BRENDA (http://www.brenda-enzymes.info/index.php4).
Review Trends in Biotechnology Vol.27 No.4
the oxidation–reduction reaction occurs in two coppercentres: electrons are transferred from the substrate tothe type 1 blue copper centre (T1,mononuclear) and then tothe trinuclear cluster (one type 2 [T2] and two type 3 [T3]copper atoms) [28]. In phsA, the oxidative coupling of two 2-aminophenol molecules to form the phenoxazinone chro-mophore involves a six-electron oxidation thought to takeplace in a series of three two-electron oxidations (seeFigure 1a), which is unique for an MCO [5,26,31].
The 3D structure of blue MCOs typically consists ofthree domains that all have a similar Greek key b-barrelstructure or the cuppredoxin fold. The three domains eachcontribute towards the formation of the active site: themononuclear site (T1) is located in domain 3, the trinuclearsite (T2 and T3) is located between domains 1 and 3, andthe hydrophobic pocket for the reducing substrate is
located between domains 2 and 3. It is the copper at theT1 site that accepts electrons from a substrate; the elec-trons are transferred along a Cys-His pathway to thetrinuclear site, where the reduction of dioxygen to wateroccurs [32]. Compared with other prokaryotic MCOs, phsAis considerably larger: the protein sequence consists of 612amino acid residues compared with the 488 of Escherichiacoli CueO (copper-resistance protein) and the 513 of Bacil-lus subtilisCotA (spore coat laccase). The additional aminoacid residues do not seem to affect the overall 3D structureof the subunits, particularly when compared to similarMCOs, such as the CueO and CotA [5].
In the early studies on the function of phsA, it wasdetermined that the protein exists in two distinct oligo-meric forms: dimers are produced by young culturesand have low catalytic activities, whereas older cultures
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produce protein hexamers that exhibit high catalyticactivity and are more stable due to the increased numberof contacts between the monomeric structures [15,33].Smith et al. [5] used X-ray diffraction in the determinationof the structure of the hexameric form. The hexameric ringstructure is centred on a pseudo sixfold axis and has a largecentral cavity (diameter of 50 A). This unique structurewas found to be stabilized by a long loop that connects twodomains between subunits that are in turn stabilized bythe presence of a fifth copper atom (type 2). This fifthcopper atom is coordinated by three histidine residues,H434, H438 and H440 [5]. Spectroscopic studies showed thateach subunit within the hexameric structure containsthese five copper atoms [8]. Furthermore, from the 3Dstructure, it was found that the substrate binding pocketand solvent channels are more similar to the laccase fromthe fungus Coprinus cinereus than to any other MCO [5].
GriF from Streptomyces griseus subsp. griseus as a type
3 copper oxidase
In contrast to S. antibioticus phsA, the amino acidsequence analyses of S. griseus subsp. griseus GriE andGriF showed an approximate 50% sequence identity to thestreptomycete MelC1 apotyrosinase (tyrosinase activator)and MelC2 tyrosinase, respectively [3], and GriF, with itshigh sequence similarity to tyrosinase, is classified as atype 3 copper oxidase (confirmed by protein BLASTanalysis [30]). The GriE protein seems to have a similarrole to the copper chaperone found in streptomycete tyr-osinases (MelC1), whereas the GriF protein is a tyrosinasehomologue. The copper-binding sites CuA and CuB, whichare present in all tyrosinases [27,34], are characterized byhistidine-rich motifs and constitute the catalytic active siteof the enzyme. The histidine residues involved in thebinding of the copper ions in GriF have been identified:His39, His58 and His67 for the CuA binding site and His222,His226 and His248 for the CuB binding site, confirming theclassification of GriF as a type 3 copper oxidase [3].
Although there is such a high sequence similarity be-tween GriF and a tyrosinase, GriF is unable to catalyseortho-hydroxylation reactions (monophenolase reactions).It has been speculated that this inability to exhibit mono-phenolase activity is linked to the presence of a ‘bulky’ Tyrresidue (absent in tyrosinases), whose phenyl ring pro-trudes into the catalytic site, thereby preventing accessof the ortho-protons of phenolic compounds to the catalyticcentre [3]. A similar structural effect is found in catecholoxidases, where the amino acid residue is a Phe residue.Furthermore, GriF has unique substrate specificity for o-aminophenols, preferring o-aminophenols above catecholssuch as 3,4-dihydroxy-L-phenylalanine (L-DOPA), a sub-strate of tyrosinase. The ability to oxidize o-aminophenolsis further limited to molecules with a phenolic hydroxylgroup in the para-position [3].
Phylogenetic analysis (M. Le Roes-Hill and S.G. Burton,unpublished observations) has confirmed the phylogeneticrelationship of GriF with tyrosinase, whereas the phsAfrom S. antibioticus has been grouped within a clade ofMCOs involved in either spore coat production or sporepigmentation, which supports the suggestion that thisenzyme could be involved in spore coat production or spore
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pigmentation inS. antibioticus [4] (Figure 2). This is a clearexample of convergent evolution, where two enzymes haveevolved as two distinct types of copper oxidases, fulfillingdifferent functions in nature but still having the ability tocatalyse similar reactions. The high bootstrap values at thenodes (Figure 2) reflect the relatedness of the MCOs andtyrosinases, further indicating the close evolutionary relat-edness of the copper oxidases, proteins that have evolved tobind copper and oxidize dioxygen to water.
The blueMCOs have the ability to catalyse a wide rangeof enzymatic reactions, suggesting variability in active siteand substrate-specificity, but there are four short regionscommon to all their protein structures. These regions havea high degree of amino acid similarity characterized byhistidine-richmotifs for the binding of copper [35]. Primaryamino acid sequence comparisons of members of the MCOfamily show the conservation of these binding domains,which also supports the classification of phsA as a blueMCO, whereas GriF typically lacks the type 1 or bluecopper binding site but has instead the required CuA
and CuB binding sites observed for type 3 copper oxidases[3,8] (see online supplementary Figure S1).
Regulation of PHS expressionPhsA from Streptomyces antibioticus
The expression of phsA from S. antibioticus is regulated atboth the transcriptional and post-transcriptional levels,which is reflected in the production of the enzyme inyoung cultures during fermentation. This is due to a highproduction of phsA mRNA and the apparent increase inspecific activity of the enzyme caused by its increasedstability compared with total cell proteins [36]. PhsAmRNA production is repressed in the presence of glucose,but unlike other catabolite repressed operons, cAMP doesnot have a role in this regulatory process. Studies by Kellyet al. [37] and Hoyt and Jones [38] further showed thatguanosine tetraphosphate (ppGpp), which is known tohave a key role during the stringent response seen inbacteria during the onset of carbon and energy starvation,might play an essential part in the regulation of phsAexpression. The discovery that phsA does not have a role inactinomycin production prompted Jones to ask two keyquestions [4]: (i) what is the function of phsA in S. anti-bioticus?; and (ii) if phsA is not involved in actinomycinproduction, how is the antibiotic produced? To answer thefirst question, Jones [4] considered the amino acidsequence of phsA and found it to be similar to CotA, aspore coat protein in Bacillus subtilis, suggesting thatphsA might have a role in spore pigment production orspore morphogenesis. Preliminary studies have confirmedthis hypothesis. For the second question, Jones [4]suggested two possibilities: (i) there is another enzymeinvolved in the process of chromophore production; or (ii)chromophore production occurs non-enzymatically underthe influence of pH and divalent cations (neither of whichhave been confirmed).
GriF from Streptomyces griseus subsp. griseus
The biosynthetic gene cluster of grixazone consists of13 genes. The promoter of griR, a pathway-specific tran-scriptional activator, is under the control of A-factor
Figure 2. Phylogenetic analysis of the DNA sequences of the most closely related sequences to GriF from Streptomyces griseus subsp. griseus and phsA from
Streptomyces antibioticus, clearly showing the high degree of relatedness of the two groups (multicopper oxidases and tyrosinases), as reflected in the high bootstrap
values at the nodes. GenBank sequence accession numbers are given in parentheses and the tree was constructed by M. Le Roes-Hill (unpublished) using the software
package MEGA 3.1.
Review Trends in Biotechnology Vol.27 No.4
(2-S-isocapryloyl-3-R-hydroxymethyl-g-butyrolactone) andphosphate depletion [39–41]. A-factor acts like a microbialhormone, controlling cellular differentiation and secondarymetabolite production in S. griseus [40]. Apart from thegenes involved in grixazone production, A-factor also con-trols the expression of type III polyketide synthases andcytochrome P450 [41] (for a more detailed discussion onA-factor, see Refs [40] and [41]).
Within the grixazone biosynthetic gene cluster, the geneproducts GriI andGriH are responsible for the formation of3-amino-4-hydroxybenzoic acid (3,4-AHBA) in a shikimatepathway-independent manner by a novel benzene ringbiosynthesis process [42]. GriE and GriF are responsiblefor the production of the phenoxazinone chromophore.GriE acts as a copper chaperone to activate GriF in asimilar way to how MelC1 activates MelC2 tyrosinase[3]. Studies have shown that a deletion of GriE and GriFresults in the accumulation of 3,4-AHBAL (3-amino-4-hydroxybenzaldehyde), the inferred substrate of GriF.Suzuki et al. [43] propose that GriC and GriD reduce3,4-AHBA to 3,4-AHBAL in an ATP-dependent manner.GriF oxidizes 3,4-AHBAL to an o-quinone imine deriva-tive, which then couples to another o-quinone imine in anon-enzymatic reaction to form the phenoxazinone [40,41](see Figure 1b). Other gene products of the grixazonebiosynthesis cluster, GriA and GriB, are speculated tobe involved in the transport of grixazone, whereas theproducts of GriS and GriT are thought to be regulators(functions still need to be determined) [41].
Phenoxazinone antibioticsTo date, there has been little research focused on deter-mining the distribution of phenoxazinone antibiotics or thePHS enzyme in nature. The most extensively studiedphenoxazinone antibiotic is the chromopeptide antibioticactinomycin (Figure 3a), produced by S. antibioticus, andits derivatives (other phenoxazinone antibiotics that havebeen isolated from actinobacteria are shown in Figure 3b).Gerber and Lechevalier [44] detected phenoxazinones instrains belonging to the genusMicrobispora (N-acetylques-tiomycin A and 2-aminophenoxazin-3-one) and later, Ger-ber [45] reported similar compounds in members of theNocardiaceae family (2-amino-1-carboxy-3H-phenoxazin-3-one). Gerber [46] also reported the discovery of twophenoxazinone compounds exhibiting antibiotic activityfrom a Streptomyces thioluteus strain (N-acetylquestiomy-cin A and 2-ethanolamino-3H-phenoxazin-3-one). The anti-bacterial and cytotoxic antibiotic glucosylquestiomycinwas isolated from Microbispora sp. strain TP-A0184 byIgarashi et al. [47], and Maskey et al. [48] isolated theanticancer agents chrandrananimycins A–C from an Acti-nomadura sp. isolate M048 as part of their screeningprogram for bioactive compounds from marine actinobac-teria. More recently, Liu et al. [49] described the discoveryof three phenoxazinone antibiotics from Streptomonosporasalina YIM 90002T (2-aminophenoxazin-3-one, N-acetyl-questiomycin and 2-methylamino-3H-phenoxazin-3-one).
The grixazones produced by S. griseus subsp. griseusdiffer slightly from the abovementioned phenoxazinones.
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Figure 3. Naturally produced phenoxazinone antibiotics isolated from actinobacterial cultures: (a) actinomycin D produced by Streptomyces antibioticus [9] and (b)
structurally related phenoxazinone antibiotics produced by Microbispora spp. [44,47], members of the family Nocardiaceae [45], Streptomyces spp. [3,46,50,51], an
Actinomadura isolate [48] and Streptomonospora salina [49].
Review Trends in Biotechnology Vol.27 No.4
Grixazone A has an aldehyde at position 8 and grixazone Bhas a carboxyl group (Figure 3b). Suzuki et al. [3] proposedthat the following antibiotics, which have a similar struc-ture to grixazone, might also require a PHS for theirsynthesis: michigazone produced by Streptomyces michi-ganensis, texazone produced by Streptomyces sp. WRAT-210, exfoliazone produced by Streptomyces exfoliatus and4-demethoxymichigazone produced by Streptomyces hal-stedii. The recently described elloxazinones produced byStreptomyces griseus ACTA 2871 [50] can also be includedin this group of antibiotics (Figure 3b). Interestingly, grix-azone B was described as early as 1989 by Zeeck et al. [51]in a patent describing the pharmacological use of phenox-azinones produced by a Streptomyces sp. strain DSM 3813.This strain also produced a variety of other phenoxazi-nones with antifungal, antiviral, antihelminthic andlipooxygenase inhibitory activity; only 15 years after thepatent was filed, Ohnishi et al. [39] described grixazone Bfrom a S. griseus subsp. griseus strain, prompting theextensive research by Suzuki et al. [3,42,43] and raisingnew interest in the production and distribution of phenox-azinone antibiotics.
Phenoxazinone antibiotics have also been detected inother microorganisms, such as the fungal species Lepiota
254
americana [52] and a Chaetosphaeria sp. [53]; as well asa marine Halomonas sp. strain GWS-BW-H8hM [54].Phenoxazinones have also been biosynthesized (usingbovine haemoglobin) or chemically synthesized and wereshown to have anticancer or cytotoxic acitivity, vasore-laxing effects, antibacterial and antiviral activity [55],immunosuppressive activity [56] and the ability to dis-rupt lipid membranes [57]. According to Shimizu et al.[58], phenoxazines that have been synthesized chemi-cally typically have low water solubility and thereforeexhibit little or no antitumour activity when comparedwith natural water soluble phenoxazinones; for example,when biosynthesized using bovine haemoglobin and o-aminophenol derivatives, the products are moresoluble in water than the synthetic compounds. Further-more, analogues of natural compounds are typicallysynthesized via low-yield, multi-step routes that areunsuitable for scale up to produce sufficient quantitiesfor clinical trials [59]. GriF and phsA have the ability tocatalyse the formation of the phenoxazinone core struc-ture in one concerted reaction and therefore could poten-tially be used as biocatalysts in preference to chemicalsynthesis in the production of novel phenoxazinone com-pounds.
Review Trends in Biotechnology Vol.27 No.4
Applications in antibiotic productionMost of the antibiotics currently available on the marketare natural products derived from actinobacteria or fungi[60]. The discovery of streptomycin in the 1940 s resultedin an extensive screening of actinobacteria, which in turnhas led to the discovery of numerous important antibiotics.However, recent decreases in screening efficiencies andincreased antibiotic resistance in pathogenic strains,particularly in multiple-drug-resistant bacteria, are incen-tives to continue screening for new antibiotics [61].
New strategies for antibiotic production continue to bedeveloped, involving culture-dependent and non-culture-dependent techniques. Miniaturized fermentation andhigh-throughput screening have been successfullyemployed by the pharmaceutical company Cubist Pharma-ceuticals [60], where macrodroplet beads are used as min-iature fermentation vessels, allowing for the screening ofmillions of antibiotic-producing strains [60]. This approachis not yet fully developed but holds great promise. Theselective isolation of ‘rare actinobacteria’ from uniqueenvironments, such as shallow marine soil, deep-sea mar-ine soil and marine organisms, has resulted in the dis-covery of a wide range of novel anticancer agents, forexample the cytotoxic phenoxazinones isolated from Strep-tomonospora salina [49] and the chandrananimycins A–Cfrom the marine Actinomadura sp. isolate M048 [48]. Theuse of specific enrichments and isolation conditions has akey role in the isolation of these ‘rare actinobacteria’, andthese parameters constantly need to be adapted dependingon the environment the sample is collected from [60,61]. Alimitation of these two approaches is the fact that they areboth culture-dependent and require physical isolation ofthe antibiotic-producing strain, and they are thereforeconstrained by the need for extensive optimization.
Non-culture-dependent approaches include genomemining for cryptic antibiotic biosynthesis gene clustersencoding unexpressed secondary metabolites, combinator-ial biosynthesis and the modification of antibiotic proper-ties by medicinal chemistry [60,61]. A key discovery madefrom genome sequencing and genome mining is the factthat actinobacteria have very large genomes comparedwith those of other bacteria, and 5–10% of the codingcapacity codes for the production of cryptic secondarymetabolites [60], an aspect that has received much inter-est; however, current research is limited by the number ofpublished genome sequences available. Combinatorial bio-synthesis is aimed at accelerating evolution to producenovel compounds and is often coupled with chemoenzy-matic studies; to date, most attention has been focused onpolyketide synthase (PKS) and nonribosomal peptidesynthase (NRPS) pathways. For this approach to be suc-cessful, a detailed understanding of the enzymatic pro-cesses is required (see Ref. [60] for how this method hasbeen successfully applied). The modification of the proper-ties of antibiotics by the approach of medicinal chemistry isstill considered to be in its infancy and needs to beexploited further. Ideally, all of these ideas should beintegrated via multidisciplinary approaches [60,61].
One further approach to developing new antibiotics isthe biocatalytic use of enzymes. Relatively few enzymeshave been exploited industrially [62], and their potential in
catalysing specifically targeted reactions depends onidentification of the desired reaction and the most suitableenzyme, as well as development of an efficient process [63].The metabolite coupling reaction catalysed by phsA andGriF provides an opportunity for biocatalytic applicationbecause they can, potentially, couple aminophenol deriva-tives with a wide range of different substituents, yielding abroad range of new phenoxazinone derivatives.
Two key questions that would have to be answered first[63] are: (i) what are the limitations in applying thesePHSs as biocatalysts?; and (ii) what would be requiredto overcome these limitations? One of the factors thatwould need to be considered is whether to use the isolatedenzyme (probably immobilized) or to make use of wholecells. The use of the isolated enzyme in the case of phsAfrom S. antibioticus would be feasible, whereas the use ofGriF from S. griseus subsp. griseus might be more proble-matic (because it is part of a complex regulatory process),especially if the genes are cloned into amore robust host foroverexpression of the protein. Cloning technologies foractinobacteria are well-defined, especially for the genusStreptomyces, and are therefore feasible [64]. Furthermore,the enzymes only require the presence of Cu2+ as a co-factor (often provided in the growth medium or in thebuffer) and require molecular oxygen to be present as anelectron acceptor, aspects that would simplify the biopro-cess design. In addition, phsA from S. antibioticus has amuch wider substrate specificity than GriF (which is lim-ited to o-aminophenols with a phenolic group in the para-position [3]), although in the latter case, this could beovercome by improving the enzyme selectivity using mol-ecular genetic techniques (see Ref. [63] for more infor-mation). PhsA from S. antibioticus, with its more variedsubstrate specificity, would therefore be a more suitablebiocatalyst than GriF, which is limited in the substrates itis able to oxidize.
The potency of new phenoxazinone products as anti-biotics also needs to be considered. The phenoxazinone corestructure has the ability to intercalate DNA betweenspecific base pairs (50-GC-30). The 2-amino group isinvolved in hydrogen bonding to the O40 and or O50 ofthe cytosine C5 residue and therefore plays a key part insuccessful intercalation, a property that has been exploitedby Bolognese et al. [65]. Keeping this in mind, o-amino-phenol derivatives with varied functional groups at the 2-amino position could be explored as substrates in thebiocatalysis of novel phenoxazinones. A chemoenzymaticapproachmight further broaden the range of final productspossible.
Combinatorial biosynthetic approaches (i.e. the manip-ulation of whole biosynthetic clusters) could potentiallygenerate large libraries of ‘novel’ antibiotics [66], althoughthe barrier of antibiotic resistance genes must be overcome[67]. The elucidation of the grixazone pathway mightfacilitate this approach in generating new phenoxazinoneantibiotics, an approach that is feasible but limited bythe lack of information on the biosynthetic pathways orprocesses involved in the production of closely relatedcompounds (michagazone, texazone, exfoliazone,demethoxymichigazone and elloxazinone). Genomesequencing has already shown that although the industrial
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strain Rhodococcus jostii strain RHA1 has not been shownto produce antibiotics, its genome encodes for severaloxidoreductases, among which is a putative PHS [68].The sequencing of more actinobacterial genomes wouldgreatly assist in determining the true distribution of theenzyme in this diverse group of bacteria.
In conclusionThis review has revealed two key areas of interest: first,the name PHS has been used to describe enzymes whichare now shown to differ significantly. Second, enzymescapable of catalysing the phenoxazinone synthetic reactionhave important potential in biocatalytic applications forproducing new antibiotics.
The classification of a small number of enzymes as PHSshas been based purely on their ability to catalyse a specificreaction, even though there are a range of other enzymesthat have the same ability. The extensive informationavailable on the two bacterial PHSs (phsA from S. anti-bioticus and GriF from S. griseus subsp. griseus) hasallowed detailed comparison of two distinct enzymes thatare both classified as a PHS (EC 1.10.3.4). However, phsAfrom S. antibioticus shares genetic and structural sim-ilarities to members of the MCO group, notably laccase,whereas GriF from S. griseus subsp. griseus shares geneticand structural similarity to the type 3 copper protein,tyrosinase. New questions arise from this information:can both these distinct enzymes be classified as PHSs(EC 1.10.3.4)? Furthermore, it might be suggested thatphsA from S. antibioticus is a laccase and GriF from S.griseus subsp. griseus is a tyrosinase. Alternatively, boththe enzymes might be considered to be PHSs, but simplysub-classes within the PHS group of enzymes. However,sequence information suggests more strongly that S. anti-bioticus phsA and S. griseus subsp. griseus GriF haveevolved convergently to address the need for biosyntheticproduction of phenoxazinone metabolites.
Conversely, the recognition that PHS enzymesresemble laccase or tyrosinase leads us to consider syn-thesis of phenoxazinone products using oxidoreductases toconvert aminophenolic substrates or their use in the pro-duction of other antibiotics. The laccase from a Trametessp. has successfully been used in the derivatization of b-lactams and was used to couple them to derivatives of 2,5-dihydroxybenzoic acid (similar in structure to ganomycin),resulting in the production of hybrid antibiotics thatshowed novel bioactivity [69]. Taking one further stepin our thinking brings us full circle because we can nowalso consider the production of new antioxidants usingactinobacterial enzyme systems capable of peroxylradical-mediated oxidation, resembling the oxidation of3-HAA by the multicopper protein ceruloplasmin [70]. Inthe actinobacteria, oxidoreductases have a key role innatural multi-step biotransformation processes, such asantibiotic production. Many are part of biosyntheticpathways, acting either as key intermediate enzymes oras part of the post-biosynthesismodification. Among theseare the P450 monooxygenases, L-amino acid oxidase,various dehydrogenases, GriF and chloroperoxidases.All these redox enzymes are potential candidates forbiotransformation but would most probably require
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cloning and expression in a robust expression systemfor commercialization [71].
Future researchThere are still gaps in the knowledge base for the group ofenzymes classified as PHSs. The lack of genetic andstructural information on the plant and fungal PHSsprecludes a conclusive comparison of all the enzymesthus classified, and the involvement of a PHS in pigmentproduction, for instance in insects, still needs to be ver-ified. The true role of phsA in S. antibioticus still needs tobe confirmed, and it is still unclear whether the phenox-azinone core of actinomycin is produced by anotherenzyme or whether it is formed non-enzymatically. Thetrue distribution of the enzyme in nature would be diffi-cult to determine in light of the high degree of similaritybetween the phsA of S. antibioticus and laccase, and theGriF of S. griseus subsp. griseus and tyrosinase; a pro-blem that is further compounded by the fact that laccaseand tyrosinase can also catalyse the same reaction asthese PHSs, precluding the screening of culture collec-tions through enzyme assays. An increase in the numberof actinobacterial genome sequences would therefore beinvaluable for this purpose. Furthermore, it would beinteresting to determine whether a PHS is present inthe phenoxazinone antibiotic-producing strains describedin this review or whether a non-enzymatic or differentprocess is involved. The increasing need for more andbetter antibiotics should also prompt more research intothe development of a PHS biocatalytic system for novelphenoxazinone synthesis.
Supplementary dataSupplementary data associated with this article can befound, in the online version, at doi:10.1016/j.tibtech.2009.01.001.
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