Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.

l‐Galactono‐1,4‐lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l‐ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l‐galactono‐1,4‐lactone (Km = 0.17 mm, kcat = 134 s−1), l‐gulono‐1,4‐lactone (Km = 13.1 mm, kcat = 4.0 s−1) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two‐electron reduced enzyme reacts poorly with molecular oxygen (kox = 6 × 102 m−1·s−1). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl‐FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD‐binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l‐galactono‐1,4‐lactone and l‐gulono‐1,4‐lactone and released their FAD cofactor more easily than wild‐type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.

The flavodoxins from Megasphaera elsdenii, Clostridium MP, and Azotobacter vinelandii were studied by 13C, 15N, and 31P NMR techniques by using various selectivity enriched oxidized riboflavin 5'-phosphate (FMN) derivatives. It is shown that the pi electron distribution in protein-bound flavin differs from that of free flavin and depends also on the apoflavoprotein used. In the oxidized state Clostridium MP and M. elsdenii flavodoxins are very similar with respect to specific hydrogen bond interaction between FMN and the apoprotein and the electronic structure of flavin. A. vinelandii flavodoxin differs from these flavodoxins in both respects, but it also differs from Desulfovibrio vulgaris flavodoxin. The similarities between A. vinelandii and D. vulgaris flavodoxins are greater than the similarities with the other two flavodoxins. The differences in the pi electron distribution in the FMN of reduced flavodoxins from A. vinelandii and D. vulgaris are even greater, but the hydrogen bond patterns between the reduced flavins and the apoflavodoxins are very similar. In the reduced state all flavodoxins studied contain an ionized prosthetic group and the isoalloxazine ring is in a planar conformation. The results are compared with existing three-dimensional data and discussed with respect to the various possible mesomeric structures in protein-bound FMN. The results are also discussed in light of the proposed hypothesis that specific hydrogen bonding to the protein-bound flavin determines the specific biological activity of a particular flavoprotein.

BackgroundAspergillus terreus is a natural producer of itaconic acid and is currently used to produce itaconic acid on an industrial scale. The metabolic process for itaconic acid biosynthesis is very similar to the production of citric acid in Aspergillus niger. However, a key enzyme in A. niger, cis-aconitate decarboxylase, is missing. The introduction of the A. terreus cadA gene in A. niger exploits the high level of citric acid production (over 200 g per liter) and theoretically can lead to production levels of over 135 g per liter of itaconic acid in A. niger. Given the potential for higher production levels in A. niger, production of itaconic acid in this host was investigated.ResultsExpression of Aspergillus terreus cis-aconitate decarboxylase in Aspergillus niger resulted in the production of a low concentration (0.05 g/L) of itaconic acid. Overexpression of codon-optimized genes for cis-aconitate decarboxylase, a mitochondrial transporter and a plasma membrane transporter in an oxaloacetate hydrolase and glucose oxidase deficient A. niger strain led to highly increased yields and itaconic acid production titers. At these higher production titers, the effect of the mitochondrial and plasma membrane transporters was much more pronounced, with levels being 5–8 times higher than previously described.ConclusionsItaconic acid can be produced in A. niger by the introduction of the A. terreus cis-aconitate decarboxylase encoding cadA gene. This results in a low itaconic acid production level, which can be increased by codon-optimization of the cadA gene for A. niger. A second crucial requirement for efficient production of itaconic acid is the expression of the A. terreus mttA gene, encoding a putative mitochondrial transporter. Expression of this transporter results in a twenty-fold increase in the secretion of itaconic acid. Expression of the A. terreus itaconic acid cluster consisting of the cadA gene, the mttA gene and the mfsA gene results in A. niger strains that produce over twenty five-fold higher levels of itaconic acid and show a twenty-fold increase in yield compared to a strain expressing only CadA.

Several aerobic microorganisms are capable of utilizing acetophenones for their growth (16,17,(30)(31)(32). However, relatively little is known about the oxidative enzymes involved in acetophenone mineralization (45,47). The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB proceeds through the initial formation of 4-hydroxyphenyl acetate and hydroquinone (31,37,47). The latter compound is further degraded via 4-hydroxymuconic semialdehyde and maleylacetate to ␤-ketoadipate (46). We have purified HapA, the enzyme responsible for the Baeyer-Villiger oxidation of 4-hydroxyacetophenone, and expressed its gene in Escherichia coli (37). Moreover, we established that this flavin adenine dinucleotide-containing monooxygenase is useful for the production of phenols and catechols, which are valuable intermediates in the synthesis of pharmaceuticals, agricultural chemicals, and material products (36,38,48).In the accompanying paper (46), we showed that the genes encoding 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenylacetate esterase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF) belong to a gene cluster (hapCDEFGHIBA) involved in 4-hydroxyacetophenone utilization. Based on biochemical data and sequence analysis, we proposed that the function of the hapC and hapD genes is linked to the conversion of hydroquinone to 4-hydroxymuconic semialdehyde.Several ring cleavage enzymes acting on substituted hydroquinones have been described. These include intradiol dioxygenases acting on hydroxyhydroquinone (4,20,22,35,39,42,55,66) and extradiol dioxygenases that are active with (homo-)gentisate (3, 28) or chlorohydroquinone (10,44,51). However, enzymes that use hydroquinone as the physiological ring cleavage substrate have not been characterized. Here we report on the purification and properties of hydroquinone dioxygenase (HQDO) from P. fluorescens ACB. It is shown that the heterotetrameric enzyme, encoded by the hapC and hapD genes, is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The present results confirm that the hapG gene, encoding an intradiol dioxygenase (46), is not involved in 4-hydroxyacetophenone degradation. This finding has important implications for the function of related genes involved in the catabolism of other aromatic compounds.

Hybrid-cluster proteins (`prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe±4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe±2S±2O] cluster, which can attain four redox states.Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe±2S] cluster instead of a [4Fe±4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe±2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 8C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (E m NADH/NAD + couple: 2320 mV) are transferred along the [2Fe±2S] cluster of the NADH oxidoreductase (E m = ±220 mV) and the [2Fe±2S] cluster of the hybrid-cluster protein (E m = ±35 mV) to the hybrid cluster (E m = ±50, +85 and +365 mV for the three redox transitions).The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.Keywords: EPR; hybrid-cluster protein (`prismane protein'); NAD(H) oxidoreductase; nitrate regulation; redox titration.The`hybrid-cluster protein' (HCP, formerly named`prismane protein') was initially purified from species of the strictly anaerobic sulfate-reducing bacterial genus Desulfovibrio: D. vulgaris (Hildenborough) [1,2] and D. desulfuricans ATCC 27774 [3,4]. This soluble cytoplasmic protein has been extensively studied because of the unusual properties of its redox-active iron clusters. Initial characterization with EPR and Mo Èssbauer spectroscopy suggested the presence of an Fe/S cluster with magnetic properties similar to those of synthetic [6Fe±6S] model compounds (`prismane' clusters; hence the name`prismane protein' was initially proposed for this protein) [1,5].Recently, the three-dimensional structure of the D. vulgaris HCP' at 1.7 A Ê resolution was obtained by X-ray crystallography [6]. The structure reveal...

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Intermediate I1 in bacterial luciferase, formed in a reaction of luciferase with FMNH-and 02, has been reinvestigated by 13C N M R spectroscopy with 13C-enriched F M N derivatives. It is shown that the previously published spectrum of the intermediate by Ghisla et al.oxidation of long-chain aliphatic aldehydes. The reaction is accompanied by emission of light. In the course of the bioluminescence reaction, several intermediates are formed, and their involvement in the reaction is still under dispute [e.g., Lee (1985) and Ziegler & Baldwin (1981)l. However, one ' Abbreviations: NMR, nuclear magnetic resonance; TMS, tetramethylsilane; FMN, oxidized riboflavin 5'-phosphate; FMNH2 and FMNH-, two-electron-reduced riboflavin 5'-phosphate in the neutral and anionic state, respectively.