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.

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.

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.

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.

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.

Dipeptide synthesis by internal adenylation domains of a multidomain enzyme involved in nonribosomal peptide synthesis.

The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for its selective substrate recognition and activation of the substrate (yielding an acyl-O-AMP intermediate) on ATP consumption. DhbF is an NRPS involved in bacillibactin synthesis and consists of multiple domains [adenylation domain, condensation domain, peptidyl carrier protein (PCP) domain, and thioesterase domain]; DhbFA1 and DhbFA2 (here named) are "internal" adenylation domains in the multidomain enzyme DhbF. We firstly succeeded in expressing and purifying the "internal" adenylation domains DhbFA1 and DhbFA2 separately. Furthermore, we initially demonstrated dipeptide synthesis by "internal" adenylation domains. When glycine and L-cysteine were used as substrates of DhbFA1, the formation of N-glycyl-L-cysteine (Gly-Cys) was observed. Furthermore, when L-threonine and L-cysteine were used as substrates of DhbFA2, N-L-threonyl-L-cysteine (Thr-Cys) was formed. These findings showed that both adenylation domains produced dipeptides by forming a carbon-nitrogen bond comprising the carboxyl group of an amino acid and the amino group of L-cysteine, although these adenylation domains are acid-thiol ligase using 4'-phosphopantetheine (bound to the PCP domain) as a substrate. Furthermore, DhbFA1 and DhbFA2 synthesized oligopeptides as well as dipeptides.

Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase.

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt-containing NHase (Co-NHase) was reported to depend on self-subunit swapping and the activator of the Co-NHase acts as a self-subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit-fused Co-NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL-18668 and the activator of low-molecular-mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt-containing proteins. Substitution of the amino acids involved in the C-terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt-binding sites might be located at the flexible C-terminal region. The cobalt-free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit-fused NHases and confirmed the metallochaperone function of the self-subunit swapping chaperone.

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.

Identification of key residues modulating the stereoselectivity of nitrile hydratase toward rac-mandelonitrile by semi-rational engineering.

Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money-, and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac-mandelonitrile (MAN). All the four selected residues had numerous collisions with rac-MAN. Five mutants significantly shifting stereoselectivity towards (S)-MAN were obtained from site-saturation mutagenesis, one of them, at position ßPhe37, exhibiting efficient production of (S)-MAN with 96.8% eep , was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)-MAN and residue ßHis37. (R)-MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi-rational design significantly reduced the select range of hot-spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity.

Correction: New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product.

[This corrects the article DOI: 10.1371/journal.pone.0175846.].

New function of aldoxime dehydratase: Redox catalysis and the formation of an unexpected product.

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig's blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.

Amide compound synthesis by adenylation domain of bacillibactin synthetase.

The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for the selective substrate recognition and its activation (as an acyl-O-AMP intermediate) during ATP consumption. DhbE, a stand-alone adenylation domain, acts on an aromatic acid, 2,3-dihydroxybenzoic acid (DHB). This activation is the initial step of the synthesis of bacillibactin that is a high-affinity small-molecule iron chelator also termed siderophore. Subsequently, the activated DHB is transferred and attached covalently to a peptidyl carrier protein domain via a thioester bond. Adenylation domains belong to the superfamily of adenylate-forming enzymes including acetyl-CoA synthetase, acyl-CoA synthetase and firefly luciferase. We previously reported a novel N-acylation reaction for an acyl-CoA synthetase (AcsA) that originally catalyzes the formation of a thioester bond between an acid and CoA, yielding acyl-CoA. This novel reaction was also confirmed for acetyl-CoA synthetase and firefly luciferase, but not yet for an adenylation domain. Here, we for the first time demonstrated the synthesis of N-acyl-L-cysteine by a stand-alone adenylation domain, DhbE. When DHB and L-cysteine were used as substrates of DhbE, N-DHB-L-cysteine was formed. A Vmax value of 0.0156±0.0008 units mg-1 and Km values of 150±18.3 mM for L-cysteine and 0.0579±0.0260 mM for DHB were obtained in this novel reaction. Furthermore, DhbE synthesized N-benzoyl-L-cysteine when benzoic acid and L-cysteine were used as substrates. Through the N-acylation reaction of DhbE, we also succeeded in the synthesis of N-aromatic acyl compounds that have never previously been reported to be produced by this enzymatic method.

Discovery of piperonal-converting oxidase involved in the metabolism of a botanical aromatic aldehyde.

Piperonal-catabolizing microorganisms were isolated from soil, the one (strain CT39-3) exhibiting the highest activity being identified as Burkholderia sp. The piperonal-converting enzyme involved in the initial step of piperonal metabolism was purified from strain CT39-3. Gene cloning of the enzyme and a homology search revealed that the enzyme belongs to the xanthine oxidase family, which comprises molybdoenzymes containing a molybdopterin cytosine dinucleotide cofactor. We found that the piperonal-converting enzyme acts on piperonal in the presence of O2, leading to formation of piperonylic acid and H2O2. The growth of strain CT39-3 was inhibited by higher concentrations of piperonal in the culture medium. Together with this finding, the broad substrate specificity of this enzyme for various aldehydes suggests that it would play an important role in the defense mechanism against antimicrobial compounds derived from plant species.

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.

Nitrile-synthesizing enzyme: Screening, purification and characterization.

Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show ß-cyano-L-alanine (ß-CNAla) synthetic activity. ß-CNAla synthase is known to catalyze nitrile synthesis: the formation of ß-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and ß-chloro-DL-alanine. In addition, we purified ß-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5-9.0 at an optimal temperature of 40-50°C. The enzyme is specific for O-acetyl-L-serine and ß-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 µmol/min/mg.

Nitrile-synthesizing enzyme: Gene cloning, overexpression and application for the production of useful compounds.

One of the nitrile-synthesizing enzymes, ß-cyano-L-alanine synthase, catalyzes ß-cyano-L-alanine (ß-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the ß-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of ß-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine. This strategy can be used for the synthesis of labeled amino acids by using a carbon-labeled KCN as a substrate, resulting in an application for positron emission tomography.

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.

Construction of a subunit-fusion nitrile hydratase and discovery of an innovative metal ion transfer pattern.

Metallochaperones are metal-binding proteins designed to deliver the appropriate metal to a target protein. The metal is usually transferred between different proteins. In this study, we discovered that metal was transferred between the same subunit of a mutant nitrile hydratase (NHase). Various "activator proteins" mediate the trafficking of metal ions into NHases. We constructed fusion NHases by fusing the ß- and α-subunits and/or the "activator proteins" of the NHase from Pseudomonas putida. The fusion NHases exhibited higher thermostability and tolerance to high concentrations of the product amide. The mechanism of the cobalt incorporation changed from a self-subunit swapping pattern to an apoprotein-specific molecular chaperone pattern in vivo and a metallochaperone pattern in vitro. Notably, the cobalt transfer occurred between the same α-subunit in the metallochaperone pattern. These results not only demonstrated the superiority of fusion-type NHases, but also revealed an innovative metal ion transfer pattern in metalloprotein biosynthesis.

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.

Streptomyces metabolites in divergent microbial interactions.

Streptomyces and related bacteria produce a wide variety of secondary metabolites. Of these, many compounds have industrial applications, but the question of why this group of microorganism produces such various kinds of biologically active substances has not yet been clearly answered. Here, we overview the results from our studies on the novel function and role of Streptomyces metabolites. The diverged action of negative and positive influences onto the physiology of various microorganisms infers the occurrence of complex microbial interactions due to the effect of small molecules produced by Streptomyces. The interactions may serve as a basis for the constitution of biological community.