Structural analysis of actinorhodin polyketide ketoreductase: cofactor binding and substrate specificity

Aromatic polyketides are a class of natural products that include many pharmaceutically important aromatic compounds. Understanding the structure and function of PKS will provide clues to the molecular basis of polyketide biosynthesis specificity. Polyketide chain reduction by ketoreductase (KR) provides regio- and stereochemical diversity. Two cocrystal structures of actinorhodin polyketide ketoreductase (act KR) were solved to 2.3 A with either the cofactor NADP(+) or NADPH bound. The monomer fold is a highly conserved Rossmann fold. Subtle differences between structures of act KR and fatty acid KRs fine-tune the tetramer interface and substrate binding pocket. Comparisons of the NADP(+)- and NADPH-bound structures indicate that the alpha6-alpha7 loop region is highly flexible. The intricate proton-relay network in the active site leads to the proposed catalytic mechanism involving four waters, NADPH, and the active site tetrad Asn114-Ser144-Tyr157-Lys161. Acyl carrier protein and substrate docking models shed light on the molecular basis of KR regio- and stereoselectivity, as well as the differences between aromatic polyketide and fatty acid biosyntheses. Sequence comparison indicates that the above features are highly conserved among aromatic polyketide KRs. The structures of act KR provide an important step toward understanding aromatic PKS and will enhance our ability to design novel aromatic polyketide natural products with different reduction patterns.     

Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.     

Aclacinomycin 10-hydroxylase is a novel substrate-assisted hydroxylase requiring S-adenosyl-L-methionine as cofactor

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-L-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-epsilon-rhodomycin, a step in the biosynthesis of the polyketide antibiotic beta-rhodomycin. S-Adenosyl-L-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis. 18O2 experiments show that the hydroxyl group is derived from molecular oxygen. The reaction further requires thiol reagents such as glutathione or dithiothreitol. Incubation of the enzyme with substrate in the absence of reductant leads to the accumulation of an intermediate with a molecular mass consistent with a perhydroxy compound. This intermediate is turned into product upon addition of glutathione. The crystal structure of an abortive enzyme-AdoMet product ternary complex reveals large conformational changes consisting of a domain rotation leading to active site closure upon binding of the anthracycline ligand. The data suggest a mechanism where decarboxylation of the substrate results in the formation of a carbanion intermediate, which is stabilized by resonance through the aromatic ring system of the anthracycline substrate. The delocalization of the electrons is facilitated by the positive charge of the cofactor AdoMet. The activation of oxygen and formation of a hydroxyperoxide intermediate occurs in a manner similar to that observed in flavoenzymes. Aclacinomycin-10-hydroxylase is the first example of a AdoMet-dependent hydroxylation reaction, a novel function for this cofactor. The enzyme lacks methyltransferase activity due to the positioning of the AdoMet methyl group unfavorable for a SN2-type methyl transfer to the substrate.     

Crystal structure of aclacinomycin-10-hydroxylase, a S-adenosyl-L-methionine-dependent methyltransferase homolog involved in anthracycline biosynthesis in Streptomyces purpurascens

Anthracyclines are aromatic polyketide antibiotics, and several of these compounds are widely used as anti-tumor drugs in chemotherapy. Aclacinomycin-10-hydroxylase (RdmB) is one of the tailoring enzymes that modify the polyketide backbone in the biosynthesis of these metabolites. RdmB, a S-adenosyl-L-methionine-dependent methyltransferase homolog, catalyses the hydroxylation of 15-demethoxy-epsilon-rhodomycin to beta-rhodomycin, one step in rhodomycin biosynthesis in Streptomyces purpurascens. The crystal structure of RdmB, determined by multiwavelength anomalous diffraction to 2.1A resolution, reveals that the enzyme subunit has a fold similar to methyltransferases and binds S-adenosyl-L-methionine. The N-terminal domain, which consists almost exclusively of alpha-helices, is involved in dimerization. The C-terminal domain contains a typical alpha/beta nucleotide-binding fold, which binds S-adenosyl-L-methionine, and several of the residues interacting with the cofactor are conserved in O-methyltransferases. Adjacent to the S-adenosyl-L-methionine molecule there is a large cleft extending to the enzyme surface of sufficient size to bind the substrate. Analysis of the putative substrate-binding pocket suggests that there is no enzymatic group in proximity of the substrate 15-demethoxy-epsilon-rhodomycin, which could assist in proton abstraction and thus facilitate methyl transfer. The lack of a suitably positioned catalytic base might thus be one of the features responsible for the inability of the enzyme to act as a methyltransferase.     

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.     

Oxygenases without requirement for cofactors or metal ions

Mono- and dioxygenases usually depend on a transition metal or an organic cofactor to activate dioxygen, or their organic substrate, or both. This review points out that there are at least two separate families of oxygenases without any apparent requirement for cofactors or metal ions: the quinone-forming monooxygenases which are important 'tailoring enzymes' in the biosynthesis of several types of aromatic polyketide antibiotics, and the bacterial dioxygenases involved in the degradation of distinct quinoline derivatives, catalyzing the 2,4-dioxygenolytic cleavage of 3-hydroxy-4-quinolones with concomitant release of carbon monoxide. The quinone-forming monooxygenases might be useful for the modification of polyketide structures, either by using them as biocatalysts, or by employing combinatorial biosynthesis approaches. Cofactor-less oxygenases present the mechanistically intriguing problem of how dioxygen is activated for catalysis. However, the reactions catalyzed by these enzymes are poorly understood in mechanistic terms. Formation of a protein radical and a substrate-derived radical, or direct electron transfer from a deprotonated substrate to molecular oxygen to form a caged radical pair may be discussed as hypothetical mechanisms. The latter reaction route is expected for substrates that can easily donate an electron to dioxygen, and requires the ability of the enzyme to stabilize anionic intermediates. Histidine residues found to be catalytically relevant in both types of cofactor-less oxygenases might be involved in substrate deprotonation and/or electrostatic stabilization.     

Crystal structure of 3-hydroxybenzoate hydroxylase from Comamonas testosteroni has a large tunnel for substrate and oxygen access to the active site

The 3-hydroxybenzoate hydroxylase (MHBH) from Comamonas testosteroni KH122-3s is a single-component flavoprotein monooxygenase, a member of the glutathione reductase (GR) family. It catalyzes the conversion of 3-hydroxybenzoate to 3,4-dihydroxybenzoate with concomitant requirements for equimolar amounts of NADPH and molecular oxygen. The production of dihydroxy-benzenoid derivative by hydroxylation is the first step in the aerobic degradation of various phenolic compounds in soil microorganisms. To establish the structural basis for substrate recognition, the crystal structure of MHBH in complex with its substrate was determined at 1.8 A resolution. The enzyme is shown to form a physiologically active homodimer with crystallographic 2-fold symmetry, in which each subunit consists of the first two domains comprising an active site and the C-terminal domain involved in oligomerization. The protein fold of the catalytic domains and the active-site architecture, including the FAD and substrate-binding sites, are similar to those of 4-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PHHY), which are members of the GR family, providing evidence that the flavoprotein aromatic hydroxylases share similar catalytic actions for hydroxylation of the respective substrates. Structural comparison of MHBH with the homologous enzymes suggested that a large tunnel connecting the substrate-binding pocket to the protein surface serves for substrate transport in this enzyme. The internal space of the large tunnel is distinctly divided into hydrophilic and hydrophobic regions. The characteristically stratified environment in the tunnel interior and the size of the entrance would allow the enzyme to select its substrate by amphiphilic nature and molecular size. In addition, the structure of the Xe-derivative at 2.5 A resolution led to the identification of a putative oxygen-binding site adjacent to the substrate-binding pocket. The hydrophobic nature of the xenon-binding site extends to the solvent through the tunnel, suggesting that the tunnel could be involved in oxygen transport.     

Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway

Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14. We had previously reported the crystal structure and enzymological analyses of the TcmN ARO/CYC, which promotes C9-C14 first-ring cyclization. However, how C7-C12 first-ring cyclization is controlled remains unresolved. In this work, we present the 2.4 ? crystal structure of ZhuI, a C7-C12-specific first-ring ARO/CYC from the type II PKS pathway responsible for the production of the R1128 polyketides. Though ZhuI possesses a helix-grip fold shared by TcmN ARO/CYC, there are substantial differences in overall structure and pocket residue composition that may be important for directing C7-C12 (rather than C9-C14) cyclization. Docking studies and site-directed mutagenesis coupled to an in vitro activity assay demonstrate that ZhuI pocket residues R66, H109, and D146 are important for enzyme function. The ZhuI crystal structure helps visualize the structure and putative dehydratase function of the didomain ARO/CYCs from KR-containing type II PKSs. The sequence-structure-function analysis described for ZhuI elucidates the molecular mechanisms that control C7-C12 first-ring polyketide cyclization and builds a foundation for future endeavors into directing cyclization patterns for engineered biosynthesis of aromatic polyketides.     

Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide biosynthetic pathway: insights into extender unit biosynthesis

Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506. Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules. The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4%. FkbI has a similar fold to acyl-CoA dehydrogenases. Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate. This structural observation coincided the genetic experiment done by Carroll et al. J. Am. Chem. Soc., 124 (2002) 4176. Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis. A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.     

Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.

1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.     

The polyketide cyclase RemF from Streptomyces resistomycificus contains an unusual octahedral zinc binding site

RemF is a polyketide cyclase involved in the biosynthesis of the aromatic pentacyclic metabolite resistomycin in Streptomyces resistomycificus. The enzyme is a member of a structurally hitherto uncharacterized class of polyketide cyclases. The crystal structure of RemF was determined by SAD and refined to 1.2 Å resolution. The enzyme subunit shows a β-sandwich structure with a topology characteristic for the cupin fold. RemF contains a metal binding site located at the bottom of the predominantly hydrophobic active site cavity. A zinc ion is coordinated to four histidine side chains, and two water molecules in octahedral ligand sphere geometry, highly unusual for zinc binding sites in proteins.     

Structural and functional analysis of tetracenomycin F2 cyclase from Streptomyces glaucescens. A type II polyketide cyclase

Tetracenomycin F2 cyclase (tcmI gene product), catalyzes an aromatic rearrangement in the biosynthetic pathway for tetracenomycin C in Streptomyces glaucescens. The x-ray structure of this small enzyme has been determined to 1.9-A resolution together with an analysis of site-directed mutants of potential catalytic residues. The protein exhibits a dimeric betaalphabeta ferredoxin-like fold that utilizes strand swapping between subunits in its assembly. The fold is dominated by four strands of antiparallel sheet and a layer of alpha-helices, which creates a cavity that is proposed to be the active site. This type of secondary structural arrangement has been previously observed in polyketide monooxygenases and suggests an evolutionary relationship between enzymes that catalyze adjacent steps in these biosynthetic pathways. Mutational analysis of all of the obvious catalytic bases within the active site suggests that the enzyme functions to steer the chemical outcome of the cyclization rather than providing a specific catalytic group. Together, the structure and functional analysis provide insight into the structural framework necessary to perform the complex rearrangements catalyzed by this class of polyketide cyclases.     

Enzymatic formation of unnatural aromatic polyketides by chalcone synthase

Substrate specificity of recombinant chalcone synthase (CHS) from Scutellaria baicalensis (Labiatae) was investigated using chemically synthesized aromatic and aliphatic CoA esters. It was demonstrated for the first time that CHS converted benzoyl-CoA to phlorobenzophenone (2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic polyketide, phlorobenzylketone (2, 4,6-trihydroxyphenylbenzylketone), whose structure was finally confirmed by chemical synthesis. Furthermore, in agreement with earlier reports, S. baicalensis CHS also accepted aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone derivatives without formation of a new aromatic ring. It was noteworthy that both aromatic and aliphatic CoA esters were accepted in the active site of the enzyme as a starter substrate for the complex condensation reaction. The low substrate specificity of CHS thus provided further insight into the structure and function of the enzyme.     

Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP-stimulated cGMP phosphodiesterase from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2'H. Modifications at N7, C8, O3' and O5' induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold. Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3', while modifications at N2H2, N3, N7, O2'H, and O5' have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein. cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3-6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.     

Succinate complex crystal structures of the alpha-ketoglutarate-dependent dioxygenase AtsK: steric aspects of enzyme self-hydroxylation

The alkylsulfatase AtsK from Pseudomonas putida S-313 is a member of the non-heme iron(II)-alpha-ketoglutarate-dependent dioxygenase superfamily. In the initial step of their catalytic cycle, enzymes belonging to this widespread and versatile family coordinate molecular oxygen to the iron center in the active site. The subsequent decarboxylation of the cosubstrate alpha-ketoglutarate yields carbon dioxide, succinate, and a highly reactive ferryl (IV) species, which is required for substrate oxidation via a complex mechanism involving the transfer of radical species. Non-productive activation of oxygen may lead to harmful side reactions; therefore, such enzymes need an effective built-in protection mechanism. One of the ways of controlling undesired side reactions is the self-hydroxylation of an aromatic side chain, which leads to an irreversibly inactivated species. Here we describe the crystal structure of the alkylsulfatase AtsK in complexes with succinate and with Fe(II)/succinate. In the crystal structure of the AtsK-Fe(II)-succinate complex, the side chain of Tyr(168) is co-ordinated to the iron, suggesting that Tyr(168) is the target of enzyme self-hydroxylation. This is the first structural study of an Fe(II)-alpha-ketoglutarate-dependent dioxygenase that presents an aromatic side chain coordinated to the metal center, thus allowing structural insight into this protective mechanism of enzyme self-inactivation.     

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel β-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.     

Examining the role of hydrogen bonding interactions in the substrate specificity for the loading step of polyketide synthase thioesterase domains

The final step in polyketide synthase-mediated biosynthesis of macrocyclic polyketides is thioesterase (TE)-catalyzed cyclization of a linear polyketide acyl chain. TEs are highly specific in the chemistry they catalyze. Understanding the molecular basis for substrate specificity of TEs is crucial for engineering these enzymes to macrocyclize non-native linear substrates. We investigated the role of hydrogen bonding interactions in the substrate specificity of formation of an acyl-enzyme intermediate for the TE from the 6-deoxyerythronolide B biosynthetic pathway. Thirteen single site-directed mutants were constructed, via removal of side chain hydrogen bonding groups from the binding cavity. Specificity constants for four different substrates with and without hydrogen bond donors and acceptors were determined for the five active mutants. The relative magnitude of specificity constants for substrates did not change for the mutant TEs. Circular dichroism spectroscopy was used to show that the majority of the catalytically inactive mutants did not fold. Two mutations were identified that enabled mutant TEs to form a folded but catalytically inactive tertiary structure. Our data do not support a role for hydrogen bonding in mediating substrate specificity of bacterial polyketide synthase TEs. The highly conserved polar residues in the binding cavity appear to stabilize the unusual substrate channel, which passes through the enzyme. We propose that hydrophobic interactions between the binding cavity and substrate drive substrate specificity, as is seen in many protein-carbohydrate recognition events. This hypothesis is in agreement with high-resolution structural data for nonhydrolyzable acyl-enzyme intermediates from the picromycin TE.     

Pyruvate decarboxylase inactivation by interaction with substrate and molecular oxygen]

Pyruvate may promote the yeast pyruvate decarboxylase inactivation when affected by molecular oxygen. In the presence of pyruvate and O2 inactivation of enzyme increases with the initial substrate concentration increasing. pH-dependence of pyruvate decarboxylase inactivation under joint action of substrate and O2 has maximum in the region 6.9-7.5. It is suggested that the influence of pyruvate and molecular oxygen is connected with the coenzyme-substrate complex oxidation in an active site of yeast pyruvate decarboxylase on the steps preceding the release of free acetaldehyde.     

Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis

Angucyclines are aromatic polyketides produced in Streptomycetes via complex enzymatic biosynthetic pathways. PgaE and CabE from S. sp PGA64 and S. sp. H021 are two related homo-dimeric FAD and NADPH dependent aromatic hydroxylases involved in the early steps of the angucycline core modification. Here we report the three-dimensional structures of these two enzymes determined by X-ray crystallography using multiple anomalous diffraction and molecular replacement, respectively, to resolutions of 1.8 A and 2.7 A. The enzyme subunits are built up of three domains, a FAD binding domain, a domain involved in substrate binding and a C-terminal thioredoxin-like domain of unknown function. The structure analysis identifies PgaE and CabE as members of the para-hydroxybenzoate hydroxylase (pHBH) fold family of aromatic hydroxylases. In contrast to phenol hydroxylase and 3-hydroxybenzoate hydroxylase that utilize the C-terminal domain for dimer formation, this domain is not part of the subunit-subunit interface in PgaE and CabE. Instead, dimer assembly occurs through interactions of their FAD binding domains. FAD is bound non-covalently in the "in"-conformation. The active sites in the two enzymes differ significantly from those of other aromatic hydroxylases. The volumes of the active site are significantly larger, as expected in view of the voluminous tetracyclic angucycline substrates. The structures further suggest that substrate binding and catalysis may involve dynamic rearrangements of the middle domain relative to the other two domains. Site-directed mutagenesis studies of putative catalytic groups in the active site of PgaE argue against enzyme-catalyzed substrate deprotonation as a step in catalysis. This is in contrast to pHBH, where deprotonation/protonation of the substrate has been suggested as an essential part of the enzymatic mechanism.     

DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubicin. To investigate the role of DnrD in this transformation, its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyze the conversion of AAME to AK presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the AAME precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters that are important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.