Identification of dioxygenases required for Aspergillus development. Studies of products, stereochemistry, and the reaction mechanism

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-(2)H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.     

Structure and reaction mechanism of basil eugenol synthase

Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.     

A density functional investigation of the extradiol cleavage mechanism in non-heme iron catechol dioxygenases

The mechanism for extradiol cleavage in non-heme iron catechol dioxygenase was modelled theoretically via density functional theory. Based on the Fe(II)-His,His,Glu motif observed in enzymes, an active site model complex, [Fe(acetate)(imidazole)(2)(catecholate)(O(2))](-), was optimized for states with six, four and two unpaired electrons (U6, U4 and U2, respectively). The transfer of the terminal atom of the coordinated dioxygen leading to "ferryl" Fe=O intermediates spontaneously generates an extradiol epoxide. The computed barriers range from 19 kcal mol(-1) on the U6 surface to approximately 25 kcal mol(-1) on the U4 surface, with overall reaction energies of +11.6, 6.3 and 7.1 kcal mol(-1) for U6, U4 and U2, respectively. The calculations for a protonated process reveal the terminal oxygen of O(2) to be the thermodynamically favoured site but subsequent oxygen transfer to the catechol has a barrier of approximately 30-40 kcal mol(-1), depending on the spin state. Instead, protonating the acetate group gives a slightly higher energy species but a subsequent barrier on the U4 surface of only 7 kcal mol(-1) relative to the hydroperoxide complex. The overall exoergicity increases to 13 kcal mol(-1). The favoured proton-assisted pathway does not involve significant radical character and has features reminiscent of a Criegee rearrangement which involves the participation of the aromatic ring pi-orbitals in the formation of the new carbon-oxygen bond. The subsequent collapse of the epoxide, attack by the coordinated hydroxide and final product formation proceeds with an overall exoergicity of approximately 75 kcal mol(-1) on the U4 surface.     

Structure and reaction mechanism of L-rhamnulose kinase from Escherichia coli

Bacterial L-rhamnulose kinase participates in the degradation of L-rhamnose, which is ubiquitous and particularly abundant in some plants. The enzyme catalyzes the transfer of the gamma-phosphate group from ATP to the 1-hydroxyl group of L-rhamnulose. We determined the crystal structures of the substrate-free kinase and of a complex between the enzyme, ADP and L-fructose, which besides rhamnulose is also processed. According to its chainfold, the kinase belongs to the hexokinase-hsp70-actin superfamily. The closest structurally known homologue is glycerol kinase. The reported structures reveal a large conformational change on substrate binding as well as the key residues involved in catalysis. The substrates ADP and beta-L-fructose are in an ideal position to define a direct in-line phosphoryl transfer through a bipyramidal pentavalent intermediate. The enzyme contains one disulfide bridge at a position where two homologous glycerol kinases are regulated by phosphorylation and effector binding, respectively, and it has two more pairs of cysteine residues near the surface that are poised for bridging. However, identical catalytic rates were observed for the enzyme in reducing and oxidizing environments, suggesting that regulation by disulfide formation is unlikely.     

The mechanism of heme oxygenase

Major advances have been made in determining the structure of heme oxygenase and the relationship between its structure and catalytic activity. The nature of the first step in the reaction sequence, heme alpha-meso-hydroxylation, is now clear, although the mechanisms that control the alpha-regiospecificity remain elusive. Hypothetical mechanisms can be written for the steps that convert alpha-meso-hydroxyheme to biliverdin, but these mechanisms must be validated before this complex reaction sequence can be fully understood. The salient conclusion appears to be that the heme-oxygenase reaction reflects the absence of interactions that channel the reaction towards a ferryl species, rather than the presence of interactions that specifically promote heme oxidation.     

α-Ketoglutarate dependent dioxygenases: A mechanism for prolyl hydroxylase action

The enzymatic conversion of proline residues to hydroxyproline residues within collagen peptides is discussed in mechanistic terms. The chemistry of previously suggested reaction mechanisms is reviewed, and a new mechanism which is based on recently published data is proposed. The key issue in the new interpretation is the coupling of the exothermic oxidative decarboxylation of α-ketoglutaric acid to the endothermic production of an oxo-iron species, which then stereospecifically effects an aliphatic hydroxylation of the proline ring.     

Peroxynitrite reaction with heme proteins.

We have reported that low levels of peroxynitrite (PN) can cause inactivation of the heme-thiolate protein prostacyclin (PGI2)-synthase by nitration of a tyrosine residue. To prove that iron catalysis is involved we studied the interaction of PN with microperoxidase and P450nor, a heme-thiolate protein of known structure. Spectral and kinetic analyses allow to conclude on a ferryl nitrogen dioxide complex as an intermediate which decomposes in the presence of an excess of PN under formation of dioxygen, nitrite, and nitrate. This occurs in a catalytic cycle which was more efficient with P450nor than with microperoxidase. If phenol was added to the reaction mixtures of PN and the ferric complexes the ratio of hydroxylated to nitrated phenols decreased compared to the metal-free system. Phenol competed with the formation of dioxygen indicating that the ferryl intermediate was involved in both pathways. One therefore can postulate that the ferryl complex reacts with phenol to give the phenoxyradical which is nitrated in the presence of nitrogen dioxide but does not give hydroxylated products as with metal-free PN. Alternately, the ferryl nitrogen dioxide complex can oxidize a second PN molecule to the radical, *OONO, which can decompose to dioxygen and NO. The latter forms N2O3, with the remaining *NO2 radical. A third pathway consists in the isomerization to nitrate which also is catalyzed by the heme proteins since the ratio of nitrite/nitrate does not change significantly during the catalytic reaction with excess of PN. Our data explain the mechanism of nitration of PGI2-synthase, suggest a role of P450nor as a PN scavenger, and favor heme-thiolate complexes for trapping PN.     

Structure and function of enzymes in heme biosynthesis

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V‐shaped glutamyl‐tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the “Radical SAM enzyme” coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure–function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.     

Structure and catalytic mechanism of horseradish peroxidase. Regiospecific meso alkylation of the prosthetic heme group by alkylhydrazines

Horseradish peroxidase is inactivated in a time-, H2O2-, and concentration-dependent manner by phenylethyl-, ethyl-, and methylhydrazine. The pseudo- first order kinetic constants for these inactivation reactions at pH 7 are: phenylethyl (KI = 115 microM, kinact = 1.5 min-1, partition ratio = 11), ethyl (KI = 145 microM, kinact = 0.08 min-1, partition ratio = 32), and methyl (KI = 3000 microM, kinact = 0.12 min-1, partition ratio = 80). At pH 5, the constants for the phenylethyl reaction change to KI = 1540 microM and kinact = 0.86 min-1. A transient absorbance at approximately 830 nm, suggestive of an isoporphyrin intermediate, is seen during these reactions. The prosthetic heme is converted by each of the three alkylhydrazines into the corresponding delta-meso-alkylated heme. Complete inactivation of the enzymes by methyl-, ethyl-, and phenylethylhydrazine is associated with alkylation of 60-70, 70, and 90%, respectively, of the prosthetic heme groups. The absence of N-alkylation and the high specificity for the delta-meso position, even with agents as small as methylhydrazine, strengthen the proposal that electron abstraction is mediated by the heme edge rather than the ferryl oxygen of horseradish peroxidase.     

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.     

Fe(NTA)-catalyzed dioxygenation of 4-t-butylcatechol and the mechanism of non-heme ferric dioxygenases

Fe(NTA), activating the ‘substrate’ 4-t-butyl- catechole, represents a functional active centre analogue of non-heme ferric dioxygenases. A Fe(NTA) catecholate complex with monodentate catecholate is the reactive species to undergo dioxygenation.