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First published online February 1, 2008; 10.1104/pp.107.114090 Plant Physiology 146:1515-1527 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Phylogenomic and Functional Analysis of Pterin-4a-Carbinolamine Dehydratase Family (COG2154) Proteins in Plants and Microorganisms1,[W],[OA]Department of Horticultural Sciences (V.N., A.N., M.J.Z., A.D.H.) and Department of Microbiology and Cell Science (V.C.), University of Florida, Gainesville, Florida 32611; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110 (S.M.B., L.-F.L.); Department of Botany, University of Queensland, Brisbane, Queensland 4072, Australia (A.M.P., J.R.B.); and Laboratoire de Physiologie Cellulaire Végétale, CNRS/CEA/INRA/Université Joseph Fourier, CEA-Grenoble, F–38054 Grenoble cedex 9, France (K.L., S.R., F.R.)
Pterin-4a-carbinolamine dehydratases (PCDs) recycle oxidized pterin cofactors generated by aromatic amino acid hydroxylases (AAHs). PCDs are known biochemically only from animals and one bacterium, but PCD-like proteins (COG2154 in the Clusters of Orthologous Groups [COGs] database) are encoded by many plant and microbial genomes. Because these genomes often encode no AAH homologs, the annotation of their COG2154 proteins as PCDs is questionable. Moreover, some COG2154 proteins lack canonical residues that are catalytically important in mammalian PCDs. Diverse COG2154 proteins of plant, fungal, protistan, and prokaryotic origin were therefore tested for PCD activity by functional complementation in Escherichia coli, and the plant proteins were localized using green fluorescent protein fusions. Higher and lower plants proved to have two COG2154 proteins, a mitochondrial one with PCD activity and a noncanonical, plastidial one without. Phylogenetic analysis indicated that the latter is unique to plants and arose from the former early in the plant lineage. All 10 microbial COG2154 proteins tested had PCD activity; six of these came from genomes with no AAH, and six were noncanonical. The results suggested the motif [EDKH]-x(3)-H-[HN]-[PCS]-x(5,6)-[YWF]-x(9)-[HW]-x(8,15)-D as a signature for PCD activity. Organisms having a functional PCD but no AAH partner include angiosperms, yeast, and various prokaryotes. In these cases, PCD presumably has another function. An ancillary role in molybdopterin cofactor metabolism, hypothesized from phylogenomic evidence, was supported by demonstrating significantly lowered activities of two molybdoenzymes in Arabidopsis thaliana PCD knockout mutants. Besides this role, we propose that partnerless PCDs support the function of as yet unrecognized pterin-dependent enzymes.
Pterin-4a-carbinolamine dehydratase (PCD; EC 4.2.1.96) is a small protein that mediates the first of two reactions in the recycling of tetrahydropterins, the cofactors of aromatic amino hydroxylases (AAHs; Thöny et al., 2000  ). PCD catalyzes the dehydration of the pterin-4a-carbinolamine formed in the hydroxylase reaction, giving a quinonoid (q)-dihydropterin that is then reduced to the tetrahydro level by q-dihydropterin reductase (Fig. 1A
). The dehydration reaction can also occur spontaneously at a low rate (Thöny et al., 2000). PCDs are best known from mammals but have also been cloned and functionally verified from fruit flies (Seong et al., 1998), apicomplexan protists (Wang et al., 2006), and the bacterium Pseudomonas aeruginosa (Zhao et al., 1994).
PCDs have no established metabolic role beyond supporting the function of AAHs by regenerating their cofactors, and this role is substantiated by genetic evidence (Thöny et al., 1998 ; Song et al., 1999). In animals, PCD supports three different AAHs that act on Phe, Tyr, or Trp (Hufton et al., 1995), while in P. aeruginosa and related bacteria, PCD supports a single Phe hydroxylase that is induced by Phe (Nakata et al., 1979; Song et al., 1999). Phe hydroxylases in animals and P. aeruginosa serve a catabolic function, converting Phe to Tyr (Fig. 1B) that can then be degraded via the homogentisate pathway (Gu et al., 1998; Arias-Barrau et al., 2004; Moran, 2005).
Crystal structures, sequences, and site-directed mutagenesis of animal and P. aeruginosa PCDs point to a canonical catalytic motif, [DE]-x(3)-H-H-P-x(5)-[YW]-x(9)-H-x(8)-D (PROSITE syntax; Hulo et al., 2006
This classical picture of PCD distribution and function, based on very few organisms, has been challenged by the advent of hundreds of sequenced genomes. Inspection of these genomes indicates that PCD-like sequences (COG2154 in the Clusters of Orthologous Groups [COGs] database; Tatusov et al., 2003 This situation led us to conduct a comprehensive comparative genomic (phylogenomic) analysis of the COG2154 proteins of plants and microorganisms to assess their diversity and possible function. This analysis guided experiments to test the PCD activity of representative COG2154 proteins and to localize them in plants. The results established that plants have functional PCDs and strongly implied that PCDs have other metabolic roles besides supporting the function of AAHs.
Phylogenomic Analysis of COG2154 in Plants and Microbes
We first systematically analyzed the distribution of COG2154 sequences in relation to that of AAH sequences in plants, fungi, protists, and prokaryotes. COG2154 sequences were identified using the National Center for Biotechnology Information (NCBI) Conserved Domain (CD) search tool (Marchler-Bauer et al., 2005
First, COG2154 sequences of two types were found in all plant taxa examined (chlorophyte algae, mosses, gymnosperms, and angiosperms). Second, a single COG2154 sequence was found in all protists surveyed and in many but not all fungi, bacteria, and archaea. Third, COG2154 sequences are not accompanied by an AAH sequence in many organisms, including angiosperms, fungi, and a diverse array of prokaryotes. Fourth, in bacteria with both COG2154 and AAH, these genes are often adjacent and sometimes clustered with homogentisate pathway genes. Fifth, certain COG2154 sequences lack one or more of the canonical His residues or other putatively crucial residues or show atypical spacing of the latter. These points are explored below in more detail. For simplicity and clarity, hereafter in the text we designate test organisms by their genus name only.
Both types of COG2154 sequences in plants have N-terminal extensions of approximately 60 to 100 amino acids compared to Pseudomonas PCD but are otherwise clearly distinct from each other (Fig. 3A ). The first type (type 1) has an almost canonical catalytic motif, departing only in having an extra residue between the H-H-P and [YW] (i.e. Y or W) elements. This type is typically predicted by three algorithms (TargetP, Predotar, and PSORT) to be targeted to mitochondria. The second type of sequence (type 2) departs radically from the canon; most notably, the [YW] element is invariably absent, and, in all cases except Pinus, one or more of the His residues are missing. Moreover, a key feature of type 2 sequences not shared with type 1 or with any other COG2154 sequences is a conserved, approximately 45-residue domain just upstream of the core COG2154 domain (Fig. 3A). Type 2 sequences are most often predicted to be plastidial.
Phylogenetic analysis of representative COG2154 sequences from plants and other organisms placed plant type 1 and type 2 proteins in two clades that branched together, with high bootstrap values, implying a shared evolutionary history (Fig. 3B). An exception was the Chlamydomonas type 1 sequence, which did not cluster with the rest. Due to the diverged nature of COG2154 sequences, other branching patterns were poorly resolved, reflected by low bootstrap values. The close relationship between plant type 1 and 2 sequences was corroborated by a comparison of intron positions: type 1 and 2 genes were found to share two introns not found in other eukaryotes (Fig. 3A). Chlamydomonas type 1 COG2154 was again unusual because it lacked both of these introns. Taken together, the above data basically suggest that an archetypal type 2 sequence arose early within the plant lineage from a type 1 sequence and acquired a novel domain. Although all plant groups have COG2154 sequences, not all have AAH sequences. As indicated in Figure 2A, Chlamydomonas, Physcomitrella, and Pinus have proteins very like animal and bacterial AAHs (35%–45% amino acid identity), but Arabidopsis and Zea lack proteins with any detectable similarity to AAHs. Data from other plant genomes and ESTs (not shown) confirmed the generality of this pattern: nonflowering plants have AAH sequences, angiosperms do not.
COG2154 sequences were found in all protists surveyed, in ascomycete fungi, and in diverse groups of bacteria and archaea (Fig. 2A), generally as a single copy. Of 467 complete bacterial and archaeal genomes analyzed, 211 genomes (45%) encoded COG2154 proteins and represented almost all the major taxa. Many of the bacteria lacking COG2154 proteins are obligate intracellular organisms, such as Rickettsia, Coxiella, and Chlamydia (Fig. 2A) that have ceded various metabolic functions to the host. Of the 211 genomes with COG2154, fewer than one-half (90) have an AAH sequence, so that the "partnerless COG2154" situation noted above for angiosperms is very common among prokaryotes.
The 90 COG2154 genes that co-occur with AAH can reasonably be presumed a priori to encode active PCDs. Moreover, as in Pseudomonas (Zhao et al., 1994 Given the strong probability of PCD activity that is implied by association evidence (i.e. co-occurrence and clustering), it is noteworthy that microbial COG2154 proteins that are associated in these ways with AAH quite often lack a canonical catalytic motif (Fig. 3C). This implies that the current motif (based only on PCDs from animals and one bacterium) is too narrowly defined. If so, it would follow that the various noncanonical microbial and plant COG2154 proteins that have no AAH partner could also be active PCD enzymes.
The above issues led us to test a range of plant and prokaryote COG2154 proteins for PCD activity and, in the process, to reexamine the catalytic motif. We used a functional complementation assay in Escherichia coli (Song et al., 1999
The complementation assay uses an E. coli Tyr auxotroph. Because E. coli lacks both Phe hydroxylase and PCD but has q-dihydropterin reductase activity (Fig. 1A), Tyr prototrophy can be restored by coexpression of foreign Phe hydroxylase and PCD genes; neither gene alone suffices (Zhao et al., 1994
Among the plant proteins, all type 1 sequences (whose catalytic motif is close to canonical) had PCD activity, whereas all the type 2 sequences lacked activity (Fig. 4, top four rows). All the microbial proteins proved to be active (Fig. 4, bottom four rows). While we cannot exclude the possibility that the truncation point used for the plant type 2 proteins caused loss of activity, we consider it unlikely because this point lay well upstream of the core COG2154 domain (Fig. 3A).
To test whether the predicted organellar targeting sequences of type 1 and 2 proteins are functional in planta, selected COG2154 coding sequences were fused to GFP and expressed in Arabidopsis. Subcellular localization in protoplasts was visualized by epifluorescence or confocal laser scanning microscopy. The Arabidopsis and Pinus type 1 proteins gave many small, punctate GFP signals that did not coincide with chloroplasts, indicating a mitochondrial location (Fig. 5A
). In contrast, the Arabidopsis type 2 protein typically gave a strong GFP signal that precisely overlapped with chlorophyll fluorescence (Fig. 5B). Our results for the Arabidopsis type 2 protein agree with a brief report, based on a yellow fluorescent protein fusion, that this protein is chloroplastic (Valkai, 2004
Essentiality Data and Functional Predictions
The puzzling occurrence of COG2154 proteins with PCD activity in genomes with no AAH led us first to mine the microbial literature for data on COG2154 knockout mutants. Five reports were found, involving four organisms, three of which lack an AAH (Table I
). In no case was the COG2154 gene classified as essential, although in the fission yeast Schizosaccharomyces, which has no AAH, deletants showed defective spore wall formation (Kakihara et al., 2003
However, given that a pterin-4a-carbinolamine can be formed by chemical oxidation of a tetrahydropterin (Moore et al., 2002 ), one hypothesis is that PCD facilitates, but is not essential for, regeneration of chemically oxidized pterins. Besides AAH cofactors, another common type of pterin is the molybdenum cofactor (Moco) and its precursors (e.g. molybdopterin). Moco occurs in all life's kingdoms and is essential for about 40 enzymes, four of which occur in plants (Schwarz and Mendel, 2006). It is very sensitive to oxidation (Rajagopalan and Johnson, 1992). Moco has a ring structure that is electronically equivalent to a tetrahydropterin (Fig. 6A
) and has some chemical characteristics of tetrahydropterins as well as others due to the third ring (Enemark and Garner, 1997; Nieter Burgmayer et al., 2004). Both substrate specificity studies (Rebrin et al., 1995) and the crystal structure of PCD complexed with a substrate analog (Cronk et al., 1996) indicate that PCD recognizes the pterin ring rather than its side chains. Thus, if Moco or its precursors form 4a-carbinolamines, they could be PCD substrates. Furthermore, some genomic evidence suggests a role for COG2154 proteins in Moco metabolism. Analysis of the genomic context of COG2154 genes in bacteria with no AAH reveals one embedded in molybdopterin synthesis gene clusters in Mycobacterium species and translationally coupled to moaC (Fig. 6B). A COG2154 gene is also next to moaC in some  -proteobacteria (Fig. 6B). Indeed, COG2154 sequences in GenBank are occasionally annotated as "putative molybdopterin biosynthesis protein."
A Link between Arabidopsis PCD and Moco Metabolism To explore a connection between PCD and Moco, two independent insertional mutants in the Arabidopsis gene (At1g29810) encoding the type 1 protein were isolated and authenticated; the mutants came from the Salk and GABI-Kat collections (Fig. 7, A and B ). Both mutations were knockouts, as judged by absence of detectable At1g29810 mRNA (Fig. 7B). The expression of the At1g29810 gene in wild-type plants was examined and found to be constitutive, with levels (relative to actin) highest in seeds (Fig. 7C). Mutant homozygotes grew normally compared to wild type in soil or when cultured in vitro (not shown).
Plantlets grown in vitro were tested for the activities of the four molybdoenzymes, nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. As these enzymes lose activity in plants with Moco synthesis defects (Schwarz and Mendel, 2006 ), we reasoned that there should also be some activity loss if PCD facilitates salvage of 4a-carbinolamines formed by oxidation of Moco or its precursors. Given the normal phenotype of the mutant plants, the activity loss was predicted to be modest, mandating high precision (low variability) in measuring enzyme activities. Pilot experiments showed that nitrate reductase and sulfite oxidase activities were low and too variable (coefficient of variation among replicates up to approximately 40%) to allow detection of subtle differences. We accordingly focused on xanthine dehydrogenase and aldehyde oxidase and developed sensitive fluorometric HPLC assays for both enzymes (see "Materials and Methods" and Supplemental Figure S1). In two separate experiments, xanthine dehydrogenase activity was significantly (P < 0.05) lower in both knockout lines than in the corresponding wild-type controls, the activity loss being between 10% and 28% (Fig. 7D). In the same experiments, aldehyde oxidase activity was also consistently lower in the knockout lines (by 17%–36%), the differences being statistically significant (P < 0.05 or <0.01) in all but one case (Fig. 7D).
The results of in vivo tests of COG2154 sequences from plants and diverse microorganisms (Fig. 4; Wang et al., 2006 ) allow definition of a new catalytic motif that can be used as a signature for PCD activity in any organism: [EDKH]-x(3)-H-[HN]-[PCS]-x(5,6)-[YWF]-x(9)-[HW]-x(8,15)-D. This motif, which is far broader than the one based solely on mutational and structural studies of mammalian and Pseudomonas PCDs, can be used to more reliably annotate COG2154 genes from plants and other organisms. By assuming that all microbial COG2154 genes co-occurring with AAH genes (Supplemental Table S1) encode active PCDs, the motif can be broadened slightly further to [EDKHQN]-x(3)-H-[HN]-[PCS]-x(5,6)-[YWFH]-x(9)-[HW]-x(8,15)-D. Our study demonstrates that COG2154 genes specifying active PCDs occur in the absence of AAH genes in many microorganisms and in angiosperms. While this surprising situation in angiosperms could be due to an evolutionarily recent loss of AAH that has not yet been followed by loss of the redundant PCD, this explanation seems improbable in light of the prevalence of PCDs in microorganisms with no AAH. It is more plausible to suppose that PCD has at least one other function besides recycling the pterin cofactors of AAHs and that this function is common to plants, fungi, bacteria, and archaea. What could this function be?
We obtained evidence to support the intriguing scenario, suggested by phylogenomics, that PCD has a role in the metabolism of Moco or its precursors, at least in Arabidopsis. Such a role would necessarily be ancillary, not central, to Moco formation because: (1) no genes are missing from the Moco biosynthesis pathway (Schwarz and Mendel, 2006
There are several other possible functions for PCDs. First, they might facilitate recycling of chemically oxidized tetrahydropterins. However, this still leaves the question of why such hypothetical tetrahydropterins would need to be recycled if there is no AAH. A second possibility, that PCDs recycle chemically oxidized tetrahydrofolates (which have a tetrahydropterin ring), might seem attractive because one common folate, 5-methyltetrahydrofolate, is known to form a 4a-hydroxy derivative upon oxidation (Gregory, 2007
A third possibility is that genomes with a PCD but no AAH have other pterin-dependent enzymes that generate 4a-carbinolamines. We view this as probable because mammals are known to have a pterin-dependent glyceryl ether monooxygenase (Taguchi and Armarego, 1998
Our results show that plants, unlike other organisms surveyed, have two distinct types of COG2154 protein. Type 1 is canonical (according to the catalytic motif just defined), has PCD activity, and is located in mitochondria. Type 2 is unique to plants, lacks PCD activity, is localized in plastids, and apparently arose from type 1 early in plant evolution. Apart from lacking the catalytic motif, type 2 proteins have a characteristic subterminal domain that includes the motif [GE]-[DN]-[FL]-G-A-R-D-P-x(3)-E-x(4)-F-G-[DE]K (Fig. 3A), which can be used for positive identification. That the type 2 protein is not a functional PCD, yet is apparently evolved from the type 1 enzyme, implies that it has taken on a different role. The presence in type 2 proteins of the subterminal domain fits with this idea. In an exploratory Arabidopsis study, overexpressing the type 2 gene (At5g51110) had no apparent phenotypic effect, and its suppression by RNA interference resulted in a small (approximately 10%) but significant reduction in leaf pigment content and a larger reduction (approximately 30%) in chloroplast number per mesophyll cell (Plume, 2002
For the plant mitochondrial PCD to participate in pterin recycling would require the existence of a q-dihydropterin reductase (Fig. 1A), which, given the lability of dihydropterins, seems likely also to be mitochondrial. q-Dihydropterin reductases in mammals, protists, and certain bacteria belong to the large and diverse short chain dehydrogenase-reductase family (Lye et al., 2002
Bioinformatics
Microbial genomes were analyzed using the SEED database and its tools (Overbeek et al., 2005
Genomic DNA of Bacillus cereus strain NRS 248, Corynebacterium glutamicum strain 534, Silicibacter pomeroyi strain DSS-3, Streptomyces avermitilis strain MA-4680, Synechocystis sp. strain PCC 6803, and Cytophaga hutchinsonii strain NCIB 9469 was purchased from the American Type Culture Collection. For Sulfolobus solfataricus strain DSM 1617 and Saccharomyces cerevisiae strain 971/6c (a gift from M.L. Agostini Carbone, Università di Milano), DNA was extracted from cells; a S. cerevisiae colony was suspended in 0.5 mL of water and microwaved for 3 min, and lyophilized S. solfataricus cells were suspended in 0.5 mL of water and boiled for 10 min. Extracts then were centrifuged for 10 min at 10,000g. The COG2154 gene of Vibrio cholerae (GenBank accession no. NC_002506) was synthesized by GenScript, adding 5µ NdeI and 3µ KpnI sites. The COG2154 coding sequences of Synechocystis and S. cerevisiae were PCR-amplified using KOD HiFi polymerase (Novagen) and primers harboring 5µ BamHI and 3µ KpnI sites. Primer sequences for COG2154 constructs are given in Supplemental Table S2. The forward primers contained a BamHI site, a stop codon in frame with LacZ, a Shine-Dalgarno sequence, and an NdeI site that included the start codon. The amplicons were cloned between the BamHI and KpnI sites of pBluescript SK– and introduced into Escherichia coli strain DH5
For complementation tests, plant COG2154 type 1 and 2 cDNAs were truncated, using PCR to replace their N-terminal putative targeting sequences by a start codon. Primers (Supplemental Table S2) harbored NdeI and KpnI sites as above. Type 1 and 2 cDNAs were amplified from the following ESTs: GenBank numbers BAC41856 and U13619 for Arabidopsis (Arabidopsis thaliana); BU647613 and BI999118 for Chlamydomonas reinhardtii; BJ203205 and BQ039368 for Physcomitrella patens; DT635299 and CO168958 for Pinus taeda; and DN218430 and EC883040 for Zea mays.
Plasmid pJS11, consisting of the P. aeruginosa phhA cloned into pACYC177 (Song et al., 1999
Full-length cDNAs of two plant type 1 COG2154 sequences (At1g29810 and its P. taeda ortholog) were amplified using primers At1g29810-GFP-Fwd and At1g29810-GFP-Rev and PtPCD-GFP-Fwd and PtPCD-GFP-Rev, respectively (Supplemental Table S3). The PCR products were digested with SalI and NcoI and cloned in-frame upstream of the GFP sequence in the pTH2 plasmid (Niwa, 2003
The coding region of type 2 COG2154 sequence At5g51110 was cloned into pAVA393 in frame with the GFP-coding region under the control of the cauliflower mosaic virus 35S promoter. The pAVA393 plasmid is based on pAVA319 (von Arnim et al., 1998
Two T-DNA insertional mutant lines (ecotype Columbia) were identified: line 648F10 was from the GABI-Kat collection and line SALK_121474 from the Salk collection. Wild-type or homozygote mutant segregants from each line were identified by PCR screening using At1g29810 gene-specific primers located 5' and 3' of the insertion site and primers located on the T-DNA (Supplemental Table S3). The insertion sites were confirmed by sequencing the amplicons obtained from mutant homozygotes. The kanamycin resistance gene was shown by PCR with primers nptB and nptD (Supplemental Table S3) to cosegregate with the insertion, indicating the absence of insertions at other loci. Line 648F10 was shown to have duplicate T-DNA insertions at the same locus in opposite orientation (Fig. 7A). Homozygous mutants and wild-type segregants were selfed and the progeny used for experiments. For reverse transcription (RT)-PCR experiments to demonstrate absence of a functional At1g29810 mRNA in the mutant lines, RNA was prepared from plantlets 4 weeks old and treated as described below. Primers used to analyze At1g29810 expression were located in the first and last exons. These primers and those for the Actin7 gene (At5g09810) are given in Supplemental Table S3. For PCR, 30 ng of cDNA was used per reaction. After 5 min at 95°C, the reaction was carried out with 35 cycles of 95°C for 30 s, 45°C for 30 s and 72°C for 50 s, and a final step at 72°C for 10 min.
Tissues were ground in liquid N2. Total RNA from the following tissues was isolated using RNeasy plant mini kits (Qiagen): seedlings 3 or 7 d old, roots from hydroponic culture, rosette leaves from plants at 16, 30, or 42 d of age, stems and cauline leaves from plants at 42 d of age, inflorescences, and siliques in early development. Total RNA from siliques at mid or late (yellowing) stages of development and from dry seeds was extracted using a LiCl precipitation method (Vicient and Delseny, 1999
Arabidopsis seeds were surface sterilized and germinated on plates containing agar and 0.33x Murashige and Skoog medium. At 12 d, seedlings were transferred aseptically to 250-mL flasks (seven per flask) containing 100 mL of 0.33x Murashige and Skoog medium containing 10 g L–1 Suc and cultured for 17 d. Flasks were shaken at 80 rpm. Temperature was 23°C to 28°C. Daylength was 12 h; photosynthetic photon flux density was 80 µE m–2 s–1. Harvested plantlets from each flask (constituting one replicate sample) were frozen in liquid N2 and stored at –80°C.
Protein extracts for assay of xanthine dehydrogenase, aldehyde oxidase, and sulfite oxidase were prepared as follows. Samples were ground in liquid N2 and the resulting powder was extracted in ice-cold buffer (1:3, w/v) containing 100 mM potassium phosphate, pH 7.5, 0.1 mM Na2MoO4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10% (w/v) polyvinylpolypyrrolidone. Extracts were centrifuged (30,000g, 20 min, 4°C), and supernatants were fractionated with ammonium sulfate (0%–60% saturation), stirring for 30 min at 4°C. After centrifuging (40,000g, 25 min, 4°C), pellets were resuspended in 1 to 2 mL of 100 mM potassium phosphate, pH 7.5, and 0.2-mL aliquots were desalted on 1-mL Sephadex G25 spin columns equilibrated with 100 mM potassium phosphate, pH 7.5, or, for sulfite oxidase assays, 20 mM Tris-acetate, pH 8.0, 0.1 mM EDTA.
Xanthine dehydrogenase was assayed with pterin as substrate (Mest et al., 1992
Sulfite oxidase assays (100 µL) contained 20 to 50 µL of desalted extract and 0.4 mM K3Fe(CN)6, in 20 mM Tris-acetate, pH 8.0, containing 0.1 mM EDTA. Sodium sulfite (2 µL, 20 mM) was added and the reaction was monitored spectrophotometrically by following reduction of ferricyanide (extinction coefficient 1,020 M–1 cm–1) at 420 nm, correcting for the base-line rate in absence of sulfite (Garrett and Rajagopalan, 1994
Nitrate reductase activity was extracted by grinding tissue (0.1–0.5 g) in a mortar with ice-cold 100 mM HEPES-KOH, pH 7.5, 2 mM EDTA, 7 mM Cys (1:5, w/v). After centrifugation at 14,000g for 10 min at 4°C, 60-µL samples of the supernatant were immediately added to assay mixtures (700 µL final volume) containing 100 mM HEPES-KOH, pH 7.5, 2 mM EDTA, 0.2 mM NADH, and 5 mM KNO3 (Jonassen et al., 2008
The following materials are available in the online version of this article.
We thank the following organizations for providing ESTs: the University of Leeds (UK), the RIKEN Bio Resource Center (Tsukuba-shi, Japan), the Carnegie Institute (Stanford, CA), the Schnable laboratory, Iowa State University (Ames, IA), the University of Georgia (Athens, GA), the J. Craig Venter Institute (Rockville, MD), and the Arizona Genomics Institute (Tucson, AZ). Received November 27, 2007; accepted January 21, 2008; published February 1, 2008.
1 This work was supported by the U.S. Department of Energy (grant no. DE–FG02–07ER64498 to V.C. and A.D.H.), by the National Institutes of Health (grant no. AI 21903 to S.M.B.), and by an endowment from the C.V. Griffin Sr. Foundation. 
2 These authors contributed equally to the article.
3 Present address: Elsevier Ltd., The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andrew D. Hanson (adha{at}ufl.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.114090 * Corresponding author; e-mail adha{at}ufl.edu.
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ABSTRACT
RESULTS
Tyr hydroxylation reaction mediated by Phe hydroxylase, a representative tetrahydropterin-dependent AAH. In AAH reactions, the tetrahydropterin cofactor is oxidized to a 4a-carbinolamine. The carbinolamine is dehydrated via the action of PCD to give a q-dihydropterin, which is then reduced to the tetrahydro level by an NAD(P)H-linked q-dihydropterin reductase (qDHPR). The natural cofactor for AAHs is tetrahydrobiopterin in mammals (Thöny et al., 2000
, β, 
Ap) was constructed by digesting pACYC177 with ScaI and FspI to delete an internal fragment of the ampicillin-resistance gene (Ap) and religating. Electroporation was used to simultaneously transform E. coli strain JP2255 (aroF363 pheA361 pheO352 tyrA382 thi-1 strR712 lacY1 xyl-15; Zhao et al., 1994