FAD-dependent C-glycoside-metabolizing enzymes in microorganisms: Screening, characterization, and crystal structure analysis.

C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme "C-glycoside 3-oxidase" named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.

Biodegradation of the methylenedioxyphenyl group in piperine and its derivatives: discovery of a novel methylenetransferase in an actinomycete.

IMPORTANCE: Pepper is a spice that has been used worldwide since the Age of Discovery. The substance that is responsible for the spiciness in pepper is piperine, a type of alkaloid. It has never been reported how piperine is degraded by microorganisms. In this study, we discovered a bacterium in the soil that is capable of catabolizing piperine as its sole nitrogen source. Furthermore, we discovered the enzyme involved in piperine metabolism. This enzyme decomposed the methylenedioxyphenyl group, which is the common structure in various plant-derived bioactive compounds such as sesamin, piperonal, safrole, and berberin. By utilizing this enzyme, piperine can be converted into a useful antioxidant compound. The findings about previously unknown metabolic pathways in nature can lead to the discovery of new enzymes and provide methods for the enzymatic synthesis of useful compounds.

Coronatine Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Overcoming Both Stomatal and Apoplastic Defenses in Dicot and Monocot Plants.

Pseudomonas cannabina pv. alisalensis is a causative agent of bacterial blight of crucifers including cabbage, radish, and broccoli. Importantly, P. cannabina pv. alisalensis can infect not only a wide range of Brassicaceae spp. but, also, green manure crops such as oat. However, P. cannabina pv. alisalensis virulence mechanisms have not been investigated and are not fully understood. We focused on coronatine (COR) function, which is one of the well-known P. syringae pv. tomato DC3000 virulence factors, in P. cannabina pv. alisalensis infection processes on both dicot and monocot plants. Cabbage and oat plants dip-inoculated with a P. cannabina pv. alisalensis KB211 COR mutant (ΔcmaA) exhibited reduced virulence compared with P. cannabina pv. alisalensis wild type (WT). Moreover, ΔcmaA failed to reopen stomata on both cabbage and oat, suggesting that COR facilitates P. cannabina pv. alisalensis entry through stomata into both plants. Furthermore, cabbage and oat plants syringe-infiltrated with ΔcmaA also showed reduced virulence, suggesting that COR is involved in overcoming not only stomatal-based defense but also apoplastic defense. Indeed, defense-related genes, including PR1 and PR2, were highly expressed in plants inoculated with ΔcmaA compared with WT, indicating that COR suppresses defense-related genes of both cabbage and oat. Additionally, salicylic acid accumulation increases after ΔcmaA inoculation compared with WT. Taken together, COR contributes to causing disease by suppressing stomatal-based defense and apoplastic defense in both dicot and monocot plants. Here, we investigated COR functions in the interaction of P. cannabina pv. alisalensis and different host plants (dicot and monocot plants), using genetically and biochemically defined COR deletion mutants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.

Discovery of a sesamin-metabolizing microorganism and a new enzyme.

Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, Vmax = 9.3 ± 0.4 (µmol⋅min(-1)⋅mg(-1)), and kcat = 7.9 ± 0.3 s(-1) Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (-)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins.

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction.

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.

Natural low-molecular mass organic compounds with oxidase activity as organocatalysts.

Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of L-ascorbic acid and L-cysteine as substrates by analytical methods for enzymes. Our findings were as follows: (i) oxidation reactions producing H2O2 proceeded upon addition of ACT to the reaction mixture; (ii) ACT was not consumed during the reactions; and (iii) a small amount (catalytic amount) of ACT consumed an excess amount of the substrates. Even at room temperature, atmospheric pressure, and neutral pH, ACT showed catalytic activity in aqueous solution, and ACT exhibited substrate specificity in the oxidation reactions. These findings reveal ACT to be an organocatalyst. ACT is known to show antibiotic activity, but its mechanism of action remains unknown. On the basis of our results, we propose that ACT kills bacteria by catalyzing the production of toxic levels of H2O2. We also screened various other natural products of bacterial, plant, and animal origins and found that several of the compounds exhibited catalytic activity, suggesting that living organisms produce and use these compounds as biocatalysts in nature.

Development of nitrilase promoter-derived inducible vectors for Streptomyces.

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis.

Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H(2)O(2) or O(2) as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 Šof the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wild-type and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.

A new synthetic route to N-benzyl carboxamides through the reverse reaction of N-substituted formamide deformylase.

Previously, we isolated a new enzyme, N-substituted formamide deformylase, that catalyzes the hydrolysis of N-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A. 101:13726-13731, 2004, doi:10.1073/pnas.0405082101). Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizing N-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C1 to C3), were active as acid substrates for the reverse reaction. Through this reaction, N-substituted carboxamides, such as NBFA, N-benzylacetamide, and N-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates.

Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism.

Polyphenol curcumin, a yellow pigment, derived from the rhizomes of a plant (Curcuma longa Linn) is a natural antioxidant exhibiting a variety of pharmacological activities and therapeutic properties. It has long been used as a traditional medicine and as a preservative and coloring agent in foods. Here, curcumin-converting microorganisms were isolated from human feces, the one exhibiting the highest activity being identified as Escherichia coli. We are thus unique in discovering that E. coli was able to act on curcumin. The curcumin-converting enzyme was purified from E. coli and characterized. The native enzyme had a molecular mass of about 82 kDa and consisted of two identical subunits. The enzyme has a narrow substrate spectrum, preferentially acting on curcumin. The microbial metabolism of curcumin by the purified enzyme was found to comprise a two-step reduction, curcumin being converted NADPH-dependently into an intermediate product, dihydrocurcumin, and then the end product, tetrahydrocurcumin. We named this enzyme "NADPH-dependent curcumin/dihydrocurcumin reductase" (CurA). The gene (curA) encoding this enzyme was also identified. A homology search with the BLAST program revealed that a unique enzyme involved in curcumin metabolism belongs to the medium-chain dehydrogenase/reductase superfamily.

Novel isonitrile hydratase involved in isonitrile metabolism.

We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., and Kobayashi, M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13726-13731). In this study, we found an enzyme involved in the first step of isonitrile metabolism, isonitrile hydratase, that hydrates isonitrile to the corresponding N-substituted formamide. First, we investigated the optimum culture conditions for the production of isonitrile hydratase. The highest enzyme activity was obtained when A. pascens F164 was cultured in a nutrient medium containing N-benzylformamide. This Arthrobacter isonitrile hydratase was purified, characterized, and compared with Pseudomonas putida N19-2 isonitrile hydratase (InhA), which is the sole one reported at present. Arthrobacter isonitrile hydratase was found to have a molecular mass of about 530 kDa and to consist of 12 identical subunits. The apparent K(m) value for cyclohexyl isocyanide was 0.95 ± 0.05 mm. A. pascens F164 grew and exhibited the isonitrile hydratase and N-substituted formamide deformylase activities when cultured in a medium containing an isonitrile as the sole carbon and nitrogen sources. However, both enzyme activities were not observed on culture in a medium containing glycerol and (NH(4))(2)SO(4) as the sole carbon and nitrogen sources, respectively. These findings suggested that the Arthrobacter enzyme is an inducible enzyme, possibly involved in assimilation and/or detoxification of isonitrile. Moreover, gene cloning of the Arthrobacter enzyme revealed no sequence similarity between this enzyme and InhA. Comparison of their properties and features demonstrated that the two enzymes are biochemically, immunologically, and structurally different from each other. Thus, we discovered a new isonitrile hydratase named InhB.

Discovery of posttranslational maturation by self-subunit swapping.

Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of beta- and alpha-subunits encoded by the nhlBA genes, respectively. An ORF, nhlE, just downstream of nhlBA, was found to be necessary for L-NHase activation. In contrast to the cobalt-containing L-NHase (holo-L-NHase containing Cys-SO(2)(-) and Cys-SO(-) metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues). We discovered an L-NHase maturation mediator, NhlAE, consisting of NhlE and the cobalt- and oxidized cysteine-containing alpha-subunit of L-NHase. The incorporation of cobalt into L-NHase was shown to depend on the exchange of the nonmodified cobalt-free alpha-subunit of apo-L-NHase with the cobalt-containing cysteine-modified alpha-subunit of NhlAE. This is a posttranslational maturation process different from general mechanisms of metallocenter biosynthesis known so far: the unexpected behavior of a protein in a protein complex, which we named "self-subunit swapping."

Metal chaperone, NhpC, involved in the metallocenter biosynthesis of nitrile hydratase.

Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.

Multiple virulence factors regulated by AlgU contribute to the pathogenicity of Pseudomonas savastanoi pv. glycinea in soybean.

Pseudomonas savastanoi pv. glycinea (Psg) causes bacterial blight of soybean. To identify candidate virulence factors, transposon-mediated mutational analysis of Psg was carried out. We syringe-inoculated soybean leaves with Psg transposon mutants and identified 28 mutants which showed reduced virulence from 1,000 mutants screened. Next, we spray-inoculated soybean leaves with these mutants and demonstrated that the algU mutant showed significantly reduced virulence together with reduced bacterial populations in planta. Expression profiles comparison between the Psg wild-type (WT) and algU mutant in HSC broth revealed that expression of coronatine (COR)-related genes (including cmaA and corR) were down-regulated in the algU mutant compared with Psg WT. Moreover, we also showed that COR production were reduced in the algU mutant compared with WT. We also demonstrated that algD, which is related to alginate biosynthesis, showed reduced expression and biofilm formation was significantly suppressed in the algU mutant. Furthermore, hrpL also showed less expression in the algU mutant. These results indicate that AlgU plays a critical role in promoting Psg pathogenesis by regulating multiple virulence factors.

C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes.

C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.

Unique biogenesis of high-molecular mass multimeric metalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swapping maturation.

Rhodococcus rhodochrous J1 produces high- and low-molecular mass nitrile hydratases (H-NHase and L-NHase, respectively), depending on the inducer. The incorporation of cobalt into L-NHase has been found to depend on the α-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-containing mediator, NhlAE (holo-NhlAE), this novel mode of post-translational maturation having been named self-subunit swapping and NhlE having been recognized as a self-subunit swapping chaperone. We discovered an H-NHase maturation mediator, NhhAG, consisting of NhhG and the α-subunit of H-NHase. The incorporation of cobalt into H-NHase was confirmed to be dependent on self-subunit swapping. For the first time, particles larger than apo-H-NHase were observed during the swapping process via dynamic light scattering measurements, suggesting the formation of intermediate complexes. On the basis of these findings, we initially proposed a possible mechanism for self-subunit swapping. Electron paramagnetic resonance analysis demonstrated that the coordination environment of a cobalt ion in holo-NhhAG is subtly different from that in H-NHase. Cobalt is inserted into cobalt-free NhhAG (apo-NhhAG) but not into apo-H-NHase, suggesting that NhhG functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, α-subunit swapping did not occur between apo-L-NHase and holo-NhhAG or between apo-H-NHase and holo-NhlAE in vitro. These findings revealed that self-subunit swapping is a subunit-specific reaction.

Copper amine oxidases catalyze the oxidative deamination and hydrolysis of cyclic imines.

Although cyclic imines are present in various bioactive secondary metabolites, their degradative metabolism remains unknown. Here, we report that copper amine oxidases, which are important in metabolism of primary amines, catalyze a cyclic imine cleavage reaction. We isolate a microorganism (Arthrobacter sp. C-4A) which metabolizes a ß-carboline alkaloid, harmaline. The harmaline-metabolizing enzyme (HarA) purified from strain C-4A is found to be copper amine oxidase and catalyze a ring-opening reaction of cyclic imine within harmaline, besides oxidative deamination of amines. Growth experiments on strain C-4A and Western blot analysis indicate that the HarA expression is induced by harmaline. We propose a reaction mechanism of the cyclic imine cleavage by HarA containing a post-translationally-synthesized cofactor, topaquinone. Together with the above results, the finding of the same activity of copper amine oxidase from E. coli suggests that, in many living organisms, these enzymes may play crucial roles in metabolism of ubiquitous cyclic imines.

Transcriptional regulation of the nitrile hydratase gene cluster in Pseudomonas chlororaphis B23.

An enormous amount of nitrile hydratase (NHase) is inducibly produced by Pseudomonas chlororaphis B23 after addition of methacrylamide as the sole nitrogen source to a medium. The expression pattern of the P. chlororaphis B23 NHase gene cluster in response to addition of methacrylamide to the medium was investigated. Recently, we reported that the NHase gene cluster comprises seven genes (oxdA, amiA, nhpA, nhpB, nhpC, nhpS, and acsA). Sequence analysis of the 1.5-kb region upstream of the oxdA gene revealed the presence of a 936-bp open reading frame (designated nhpR), which should encode a protein with a molecular mass of 35,098. The deduced amino acid sequence of the nhpR product showed similarity to the sequences of transcriptional regulators belonging to the XylS/AraC family. Although the transcription of the eight genes (nhpR, oxdA, amiA, nhpABC, nhpS, and acsA) in the NHase gene cluster was induced significantly in the P. chlororaphis B23 wild-type strain after addition of methacrylamide to the medium, transcription of these genes in the nhpR disruptant was not induced, demonstrating that nhpR codes for a positive transcriptional regulator in the NHase gene cluster. A reverse transcription-PCR experiment revealed that five genes (oxdA, amiA, nhpA, nhpB, and nhpC) are cotranscribed, as are two other genes (nhpS and acsA). The transcription start sites for nhpR, oxdA, nhpA, and nhpS were mapped by primer extension analysis, and putative -12 and -24 sigma(54)-type promoter binding sites were identified. NhpR was found to be the first transcriptional regulator of NHase belonging to the XylS/AraC family.

Hemoglobin catalyzes CoA degradation and thiol addition to flavonoids.

In the presence of CoA, cell-free extracts prepared from porcine liver was found to convert 7,8-dihydroxyflavone (DHF) to a pantetheine conjugate, which was a novel flavonoid. We purified a 7,8-DHF-converting enzyme from the extracts, and identified it as hemoglobin (Hb). The purified Hb showed the following two activities: (i) degradation of CoA into pantetheine through hydrolytic cleavage to yield pantetheine and 3'-phospho-adenosine-5'-diphosphate (ADP) independently of heme, and (ii) addition of a thiol (e.g., pantetheine, glutathione and cysteine) to 7,8-DHF through C-S bond formation. Human Hb also exhibited the above flavonoid-converting activity. In addition, heme-containing enzymes such as peroxidase and catalase added each of pantetheine, glutathione and cysteine to the flavonoid, although no pantetheine conjugates were synthesized when CoA was used as a substrate. These findings indicated that the thiol-conjugating activity is widely observed in heme-containing proteins. On the other hand, only Hb catalyzed the hydrolysis of CoA, followed by the thiol conjugation to synthesize the pantetheine conjugate. To the best of our knowledge, this is the first report showing that Hb has the catalytic ability to convert naturally occurring bioactive compounds, such as dietary flavonoids, to the corresponding conjugates in the presence of thiol donors or CoA.