Agrobacterium Tumefaciens S33 Research Articles

Rid Enhances the 6-Hydroxypseudooxynicotine Dehydrogenase Reaction in Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine through a hybrid of the pyridine and pyrrolidine pathways. The oxidation of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxypseudooxynicotine dehydrogenase (Pno) is an important step in the breakdown of the N-heterocycle in this pathway. Although Pno has been characterized, the reaction is not fully understood; what is known is that it starts at a high speed followed by a rapid drop in the reaction rate, leading to the formation of a very small amount of product. In this study, we speculated that an unstable imine intermediate that is toxic with regard to the metabolism is produced in the reaction. We found that a Rid protein (designated Rid-NC) encoded by a gene in the nicotine-degrading gene cluster enhanced the reaction. Rid is a widely distributed family of small proteins with various functions, and some subfamilies have deaminase activity to eliminate the toxicity of the reactive intermediate, imine. Biochemical analyses showed that Rid-NC relieved the toxicity of the presumed imine intermediate produced in the Pno reaction and that, in the presence of Rid-NC, Pno maintained a high level of activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene led to slower growth of strain S33 on nicotine. The mechanism of Rid-NC-mediated detoxification of the imine intermediate was discussed. A phylogenetic analysis indicated that Rid-NC belongs to the rarely studied Rid6 subfamily. These results further our understanding of the biochemical mechanism of nicotine degradation and provide new insights into the function of the Rid6 subfamily proteins.IMPORTANCE Rid is a family of proteins that participate in metabolite damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. Rid-NC hydrolyzed the presumed reactive imine intermediate produced in the reaction to remove its toxicity on Pno. The finding furthers our understanding of the metabolic process of the toxic N-heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of N-heterocyclic aromatic alkaloids, in addition to the amino acid metabolism, and that Rid6-subfamily proteins also have deaminase activity, similar to the RidA subfamily. The ability of reactive imines to damage a non-pyridoxal-5'-phosphate-dependent enzyme was reported. This study provides new insights into the function of the Rid family of proteins.

Physiology of a Hybrid Pathway for Nicotine Catabolism in Bacteria

Nicotine is a major N-heterocyclic aromatic alkaloid produced in tobacco plants and the main toxic chemical in tobacco waste. Due to its complex physiological effects and toxicity, it has become a concern both in terms of public health and the environment. A number of bacteria belonging to the genera Arthrobacter and Pseudomonas can degrade nicotine via the pyridine and pyrrollidine pathways. Recently, a novel hybrid of the pyridine and pyrrolidine pathways (also known as the VPP pathway) was found in the Rhizobiale group bacteria Agrobacterium tumefaciens S33, Shinella sp. HZN7 and Ochrobactrum sp. SJY1 as well as in other group bacteria. The special mosaic pathway has attracted much attention from microbiologists in terms of the study of their molecular and biochemical mechanisms. This will benefit the development of new biotechnologies in terms of the use of nicotine, the enzymes involved in its catabolism, and the microorganisms capable of degrading the alkaloid. In this pathway, some metabolites are hydroxylated in the pyridine ring or modified in the side chain with active groups, which can be used as precursors for the synthesis of some important compounds in the pharmaceutical and agricultural industries. Moreover, some enzymes may be used for industrial biocatalysis to transform pyridine derivatives into desired chemicals. Here, we review the molecular and biochemical basis of the hybrid nicotine-degrading pathway and discuss the electron transport in its oxidative degradation for energy conservation and bacterial growth.

Cloning and characterization the nicotine degradation enzymes 6-hydroxypseudooxynicotine amine oxidase and 6-hydroxy-3-succinoylpyridine hydroxylase in Pseudomonas geniculata N1

Microbial catabolism plays a crucial role in the removal of toxic alkaloids including nicotine from tobacco waste. Pseudomonas geniculata N1, an effective nicotine-degrader, possesses a variant of the pyridine and pyrrolidine (VPP) nicotine catabolic pathway. In this study, a 20-kbp gene cluster was found to contain eight nicotine-degrading genes. In comparison to Sphingomonas melonis TY, Ochrobactrum sp. SJY1, Shinella sp. HZN7, and Agrobacterium tumefaciens S33, these genes are tightly clustered in the P. geniculata N1 genome. The gene hisD encoding a 6-hydroxypseudooxynicotine (6-HPON) amine oxidase was heterologously expressed in Pseudomonas putida KT2440 and the recombinant strain acquired the ability to transform 6-HPON to 6-hydroxy-3-succinoylpyridine (HSP). The vD gene encodes a flavin-containing NADH-dependent monooxygenase, which converted HSP to 2,5-dihydroxypyridine was cloned in E. coli and characterized. Both genes, hisD and vD, were significantly upregulated in response to nicotine. This study expands our knowledge of the VPP nicotine catabolic pathway in bacteria.

6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

Green route to synthesis of valuable chemical 6-hydroxynicotine from nicotine in tobacco wastes using genetically engineered Agrobacterium tumefaciens S33

BackgroundTobacco is widely planted as an important nonfood economic crop throughout the world, and large amounts of tobacco wastes are generated during the tobacco manufacturing process. Tobacco and its wastes contain high nicotine content. This issue has become a major concern for health and environments due to its toxicity and complex physiological effects. The microbial transformation of nicotine into valuable functionalized pyridine compounds is a promising way to utilize tobacco and its wastes as a potential biomass resource. Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways, in which several intermediates, such as 6-hydroxynicotine, can be used as renewable precursors to synthesize drugs and insecticides. This provides an opportunity to produce valuable chemical 6-hydroxynicotine from nicotine via biocatalysis using strain S33.ResultsTo accumulate the intermediate 6-hydroxynicotine, we firstly identified the key enzyme decomposing 6-hydroxynicotine, named 6-hydroxynicotine oxidase, and then disrupted its encoding gene in A. tumefaciens S33. With the whole cells of the mutant as a biocatalyst, we tested the possibility to produce 6-hydroxynicotine from the nicotine of tobacco and its wastes and optimized the reaction conditions. At 30 °C and pH 7.0, nicotine could be efficiently transformed into 6-hydroxynicotine by the whole cells cultivated with glucose/ammonium/6-hydroxy-3-succinoylpyridine medium. The molar conversion and the specific catalytic rate reached approximately 98% and 1.01 g 6-hydroxynicotine h−1 g−1 dry cells, respectively. The product could be purified easily by dichloromethane extraction with a recovery of 76.8%, and was further confirmed by UV spectroscopy, mass spectroscopy, and NMR analysis.ConclusionsWe successfully developed a novel biocatalytic route to 6-hydroxynicotine from nicotine by blocking the nicotine catabolic pathway via gene disruption, which provides an alternative green strategy to utilize tobacco and its wastes as a biomass resource by converting nicotine into valuable hydroxylated-pyridine compounds.

Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min-1 · mg of protein-1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.

Genomic and transcriptomic analyses of Agrobacterium tumefaciens S33 reveal the molecular mechanism of a novel hybrid nicotine-degrading pathway

Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways. It can be utilized to remove nicotine from tobacco wastes and transform nicotine into important functionalized pyridine precursors for some valuable drugs and insecticides. However, the molecular mechanism of the hybrid pathway is still not completely clear. Here we report the genome analysis of strain S33 and its transcriptomes grown in glucose-ammonium medium and nicotine medium. The complete gene cluster involved in nicotine catabolism was found to be located on a genomic island composed of genes functionally similar but not in sequences to those of the pyridine and pyrrolidine pathways, as well as genes encoding plasmid partitioning and replication initiation proteins, conjugal transfer proteins and transposases. This suggests that the evolution of this hybrid pathway is not a simple fusion of the genes involved in the two pathways, but the result of a complicated lateral gene transfer. In addition, other genes potentially involved in the hybrid pathway could include those responsible for substrate sensing and transport, transcription regulation and electron transfer during nicotine degradation. This study provides new insights into the molecular mechanism of the novel hybrid pathway for nicotine degradation.

Characterization of a Novel Nicotine Degradation Gene Cluster ndp in Sphingomonas melonis TY and Its Evolutionary Analysis.

Sphingomonas melonis TY utilizes nicotine as a sole source of carbon, nitrogen, and energy through a variant of the pyridine and pyrrolidine pathways (VPP). A 31-kb novel nicotine-degrading gene cluster, ndp, in strain TY exhibited a different genetic organization with the vpp cluster in strains Ochrobactrum rhizosphaerae SJY1 and Agrobacterium tumefaciens S33. Genes in vpp were separated by a 20-kb interval sequence, while genes in ndp were localized together. Half of the homolog genes were in different locus in ndp and vpp. Moreover, there was a gene encoding putative transporter of nicotine or other critical metabolite in ndp. Among the putative nicotine-degrading related genes, the nicotine hydroxylase, 6-hydroxy-L-nicotine oxidase, 6-hydroxypseudooxynicotine oxidase, and 6-hydroxy-3-succinyl-pyridine monooxygenase responsible for catalyzing the transformation of nicotine to 2, 5-dihydropyridine in the initial four steps of the VPP were characterized. Hydroxylation at C6 of the pyridine ring and dehydrogenation at the C2–C3 bond of the pyrrolidine ring were the key common reactions in the VPP, pyrrolidine and pyridine pathways. Besides, VPP and pyrrolidine pathway shared the same latter part of metabolic pathway. After analysis of metabolic genes in the pyridine, pyrrolidine, and VPP pathways, we found that both the evolutionary features and metabolic mechanisms of the VPP were more similar to the pyrrolidine pathway. The linked ndpHFEG genes shared by the VPP and pyrrolidine pathways indicated that these two pathways might share the same origin, but variants were observed in some bacteria. And we speculated that the pyridine pathway was distributed in Gram-positive bacteria and the VPP and pyrrolidine pathways were distributed in Gram-negative bacteria by using comprehensive homologs searching and phylogenetic tree construction.

Genome sequence of the nicotine-degrading Agrobacterium tumefaciens S33

Agrobacterium tumefaciens S33 is capable of growing with nicotine as the sole source of carbon and nitrogen, and has the potential to dispose of tobacco wastes and transform nicotine into functionalized pyridines intermediates, which are important precursors for some valuable drugs and insecticides. Here we report the complete genome sequence of strain S33 and predict the gene cluster involved in nicotine catabolism according to the annotation.

Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33.

Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.

Nicotine-degrading microorganisms and their potential applications.

Nicotine-degrading microorganisms (NDMs) are a special microbial group which can use nicotine as the sole carbon and nitrogen source for growth. Since the 1950s, the bioconversion of nicotine by microbes has received increasing attention, and several NDMs have been identified, such as Arthrobacter nicotinovorans, Microsporum gypseum, Pellicularia filamentosa JTS-208, and Pseudomonas sp. 41. In recent years, increasing numbers of NDMs have been isolated and identified from tobacco plantation soil, leaf, and tobacco waste. Meanwhile, the metabolic pathway and degradation mechanism of nicotine have been elucidated in several NDMs, such as A. nicotinovorans, Agrobacterium tumefaciens S33, Aspergillus oryzae, and Pseudomonas putida S16. Moreover, several NDMs have been used in improving the quality of cigarettes, treating tobacco waste, and producing valuable intermediates of nicotine. Here, we summarize the diversity, phylogenetic analysis, and potential applications of NDMs.

6-hydroxy-3-succinoylpyridine hydroxylase catalyzes a central step of nicotine degradation in Agrobacterium tumefaciens S33.

Nicotine is a main alkaloid in tobacco and is also the primary toxic compound in tobacco wastes. It can be degraded by bacteria via either pyridine pathway or pyrrolidine pathway. Previously, a fused pathway of the pyridine pathway and the pyrrolidine pathway was proposed for nicotine degradation by Agrobacterium tumefaciens S33, in which 6-hydroxy-3-succinoylpyridine (HSP) is a key intermediate connecting the two pathways. We report here the purification and properties of an NADH-dependent HSP hydroxylase from A. tumefaciens S33. The 90-kDa homodimeric flavoprotein catalyzed the oxidative decarboxylation of HSP to 2,5-dihydroxypyridine (2,5-DHP) in the presence of NADH and FAD at pH 8.0 at a specific rate of about 18.8±1.85 µmol min−1 mg protein−1. Its gene was identified by searching the N-terminal amino acid residues of the purified protein against the genome draft of the bacterium. It encodes a protein composed of 391 amino acids with 62% identity to HSP hydroxylase (HspB) from Pseudomonas putida S16, which degrades nicotine via the pyrrolidine pathway. Considering the application potential of 2,5-DHP in agriculture and medicine, we developed a route to transform HSP into 2,5-DHP with recombinant HSP hydroxylase and an NADH-regenerating system (formate, NAD+ and formate dehydrogenase), via which around 0.53±0.03 mM 2,5-DHP was produced from 0.76±0.01 mM HSP with a molar conversion as 69.7%. This study presents the biochemical properties of the key enzyme HSP hydroxylase which is involved in the fused nicotine degradation pathway of the pyridine and pyrrolidine pathways and a new green route to biochemically synthesize functionalized 2,5-DHP.

Biodegradation of nicotine by a novel nicotine-degrading bacterium, Pseudomonas plecoglossicida TND35 and its new biotransformation intermediates

Tobacco wastes that contain nicotine alkaloids are harmful to human health and the environment. In the investigation, a novel nicotine-biodegrading bacterium TND35 was isolated and identified as Pseudomonas plecoglossicida on the basis of phenotypic, biochemical characteristics and 16S rRNA sequence homology. We have studied the nicotine biodegradation potential of strain TND35 by detecting the intermediate metabolites using an array of approaches such as HPLC, GC-MS, NMR and FT-IR. Biotransformation metabolites, N-methylmyosmine, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and other three new intermediate metabolites namely, 3,5-bis(1-methylpyrrolidin-2-yl) pyridine, 2,3-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol and 5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one have been identified. Interestingly, these intermediate metabolites suggest that the strain TND35 employs a novel nicotine biodegradation pathway, which is different from the reported pathways of Aspergillus oryzae 112822, Arthrobacter nicotinovorans pAO1, Agrobacterium tumefaciens S33 and other species of Pseudomonas. The metabolite, HPB reported in this study can also be used as biochemical marker for tobacco related cancer studies.

Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway

Nicotine, a major alkaloid in tobacco plants and the main toxic chemical in tobacco wastes, can be transformed by bacteria into hydroxylated-pyridine intermediates, which are important precursors for the chemical synthesis of valuable drugs and insecticides. Such biotransformation could be a useful approach to utilize tobacco and its wastes. In this study, we explored nicotine degradation by a recently isolated Agrobacterium tumefaciens S33 by identifying the intermediates during its growth on nicotine and during transformation of nicotine with its resting cells. Five hydroxylated-pyridine intermediates were detected through multiple approaches, including GC-HR-MS, HPLC, and ESI-Q-TOF MS analyses. Surprisingly, these identified intermediates suggest that strain S33 employs a novel pathway that is different from the two characterized pathways described in Arthrobacter and Pseudomonas. Based on these findings, we propose that strain S33 is able to transform nicotine to 6-hydroxy-pseudooxynicotine first via the pyridine pathway through 6-hydroxy-L-nicotine and 6-hydroxy-N-methylmyosmine, and then, it turns to the pyrrolidine pathway with the formation of 6-hydroxy-3-succinoylpyridine and 2,5-dihydroxypyridine. The activities of the key enzymes, nicotine dehydrogenase, 6-hydroxy-L-nicotine oxidase, and 6-hydroxy-3-succinoylpyridine hydroxylase, were demonstrated in the cell extract of strain S33 and by partially enriched enzymes. Moreover, the cell extract could transform 6-hydroxy-pseudooxynicotine into 6-hydroxy-3-succinoylpyridine by coupling with 6-hydroxy-L-nicotine oxidation reaction by 6-hydroxy-L-nicotine oxidase. These results indicated that strain S33 can transform nicotine into renewable hydroxylated-pyridine intermediates by the special pathway, in which at least three intermediates, 6-hydroxy-L-nicotine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine, have potential to be further chemically modified into useful compounds.