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Functional Identification of a Novel Gene, moaE, for 3-Succinoylpyridine Degradation in Pseudomonas putida S16.

Jiang Y, Tang H, Wu G, Xu P - Sci Rep (2015)

Bottom Line: Our previous work revealed that the heterotrimeric enzyme (SpmA, SpmB, and SpmC) requires molybdopterin cytosine dinucleotide as a cofactor for their activities.In this study, we knocked out four genes, including PPS_1556, PPS_2936, PPS_4063, and PPS_4397, and found that a novel gene, PPS_4397 encoding moaE, is necessary for molybdopterin cytosine dinucleotide biosynthesis.Resting cell reactions of the moaE deletion mutant incubated with 3 g l(-1) nicotine at 30 °C resulted in accumulation of 3-succinoylpyridine, and the strain complemented by the moaE gene regained the ability to convert 3-succinoylpyridine.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.

ABSTRACT
Microbial degradation of N-heterocyclic compounds, including xanthine, quinoline, nicotinate, and nicotine, frequently requires molybdenum hydroxylases. The intramolecular electron transfer chain of molybdenum hydroxylases consists of a molybdenum cofactor, two distinct [2Fe-2S] clusters, and flavin adenine dinucleotide. 3-Succinoylpyridine monooxygenase (Spm), responsible for the transformation from 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine, is a crucial enzyme in the pyrrolidine pathway of nicotine degradation in Pseudomonas. Our previous work revealed that the heterotrimeric enzyme (SpmA, SpmB, and SpmC) requires molybdopterin cytosine dinucleotide as a cofactor for their activities. In this study, we knocked out four genes, including PPS_1556, PPS_2936, PPS_4063, and PPS_4397, and found that a novel gene, PPS_4397 encoding moaE, is necessary for molybdopterin cytosine dinucleotide biosynthesis. Resting cell reactions of the moaE deletion mutant incubated with 3 g l(-1) nicotine at 30 °C resulted in accumulation of 3-succinoylpyridine, and the strain complemented by the moaE gene regained the ability to convert 3-succinoylpyridine. In addition, reverse transcription-quantitative polymerase chain reaction analysis indicated that the transcriptional levels of the genes of moaE, spmA, and spmC of Pseudomonas putida S16 were distinctly higher when grown in nicotine medium than in glycerol medium.

No MeSH data available.


Related in: MedlinePlus

3-Succinoylpyridine degradation in P. putida S16.Spm catalyzes 3-succinoylpyridine (SP) to 6-hydroxy-3-succinoylpyridine (HSP) in the pyrrolidine pathway. Spm has three subunits SpmA, SpmB, SpmC, respectively binding three cofactors Mo-MCD, FAD, and [2Fe-2S] clusters. MoaE is crucial for Mo-MCD synthesis and Spm holoenzyme activity.
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f1: 3-Succinoylpyridine degradation in P. putida S16.Spm catalyzes 3-succinoylpyridine (SP) to 6-hydroxy-3-succinoylpyridine (HSP) in the pyrrolidine pathway. Spm has three subunits SpmA, SpmB, SpmC, respectively binding three cofactors Mo-MCD, FAD, and [2Fe-2S] clusters. MoaE is crucial for Mo-MCD synthesis and Spm holoenzyme activity.

Mentions: Large quantities of tobacco wastes containing high concentration of nicotine are produced during tobacco manufacturing yearly1. Nicotine is well known to be harmful to human health and can cross biological membranes and blood-brain barrier easily2. Microorganisms such as Pseudomonas are important for degrading nicotine, and the pyrrolidine pathway of nicotine degradation has been systematically unraveled in Pseudomonas putida strain S163456. The pyrrolidine ring is first dehydrogenated to N-methylmyosmine, followed by spontaneous hydrolysis and oxidation to form 3-succinoylpyridine (SP). The molybdenum-dependent enzyme 3-succinoylpyridine monooxygenase (Spm) catalyzes SP to 6-hydroxy-3-succinoylpyridine (HSP) (Fig. 1). This enzyme is composed of three subunits: a large subunit carrying molybdopterin cytosine dinucleotide (Mo-MCD), a middle subunit binding a flavin adenine dinucleotide (FAD) molecule, and a small subunit with two [2Fe-2S] clusters3. When the spmABC genes are expressed in Escherichia coli, an inactive Spm is formed that lacks the Mo-MCD cofactor. Instead, the cofactor in E. coli is molybdopterin guanine dinucleotide (MGD), which cannot be integrated into the functional apoprotein Spm3.


Functional Identification of a Novel Gene, moaE, for 3-Succinoylpyridine Degradation in Pseudomonas putida S16.

Jiang Y, Tang H, Wu G, Xu P - Sci Rep (2015)

3-Succinoylpyridine degradation in P. putida S16.Spm catalyzes 3-succinoylpyridine (SP) to 6-hydroxy-3-succinoylpyridine (HSP) in the pyrrolidine pathway. Spm has three subunits SpmA, SpmB, SpmC, respectively binding three cofactors Mo-MCD, FAD, and [2Fe-2S] clusters. MoaE is crucial for Mo-MCD synthesis and Spm holoenzyme activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548258&req=5

f1: 3-Succinoylpyridine degradation in P. putida S16.Spm catalyzes 3-succinoylpyridine (SP) to 6-hydroxy-3-succinoylpyridine (HSP) in the pyrrolidine pathway. Spm has three subunits SpmA, SpmB, SpmC, respectively binding three cofactors Mo-MCD, FAD, and [2Fe-2S] clusters. MoaE is crucial for Mo-MCD synthesis and Spm holoenzyme activity.
Mentions: Large quantities of tobacco wastes containing high concentration of nicotine are produced during tobacco manufacturing yearly1. Nicotine is well known to be harmful to human health and can cross biological membranes and blood-brain barrier easily2. Microorganisms such as Pseudomonas are important for degrading nicotine, and the pyrrolidine pathway of nicotine degradation has been systematically unraveled in Pseudomonas putida strain S163456. The pyrrolidine ring is first dehydrogenated to N-methylmyosmine, followed by spontaneous hydrolysis and oxidation to form 3-succinoylpyridine (SP). The molybdenum-dependent enzyme 3-succinoylpyridine monooxygenase (Spm) catalyzes SP to 6-hydroxy-3-succinoylpyridine (HSP) (Fig. 1). This enzyme is composed of three subunits: a large subunit carrying molybdopterin cytosine dinucleotide (Mo-MCD), a middle subunit binding a flavin adenine dinucleotide (FAD) molecule, and a small subunit with two [2Fe-2S] clusters3. When the spmABC genes are expressed in Escherichia coli, an inactive Spm is formed that lacks the Mo-MCD cofactor. Instead, the cofactor in E. coli is molybdopterin guanine dinucleotide (MGD), which cannot be integrated into the functional apoprotein Spm3.

Bottom Line: Our previous work revealed that the heterotrimeric enzyme (SpmA, SpmB, and SpmC) requires molybdopterin cytosine dinucleotide as a cofactor for their activities.In this study, we knocked out four genes, including PPS_1556, PPS_2936, PPS_4063, and PPS_4397, and found that a novel gene, PPS_4397 encoding moaE, is necessary for molybdopterin cytosine dinucleotide biosynthesis.Resting cell reactions of the moaE deletion mutant incubated with 3 g l(-1) nicotine at 30 °C resulted in accumulation of 3-succinoylpyridine, and the strain complemented by the moaE gene regained the ability to convert 3-succinoylpyridine.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.

ABSTRACT
Microbial degradation of N-heterocyclic compounds, including xanthine, quinoline, nicotinate, and nicotine, frequently requires molybdenum hydroxylases. The intramolecular electron transfer chain of molybdenum hydroxylases consists of a molybdenum cofactor, two distinct [2Fe-2S] clusters, and flavin adenine dinucleotide. 3-Succinoylpyridine monooxygenase (Spm), responsible for the transformation from 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine, is a crucial enzyme in the pyrrolidine pathway of nicotine degradation in Pseudomonas. Our previous work revealed that the heterotrimeric enzyme (SpmA, SpmB, and SpmC) requires molybdopterin cytosine dinucleotide as a cofactor for their activities. In this study, we knocked out four genes, including PPS_1556, PPS_2936, PPS_4063, and PPS_4397, and found that a novel gene, PPS_4397 encoding moaE, is necessary for molybdopterin cytosine dinucleotide biosynthesis. Resting cell reactions of the moaE deletion mutant incubated with 3 g l(-1) nicotine at 30 °C resulted in accumulation of 3-succinoylpyridine, and the strain complemented by the moaE gene regained the ability to convert 3-succinoylpyridine. In addition, reverse transcription-quantitative polymerase chain reaction analysis indicated that the transcriptional levels of the genes of moaE, spmA, and spmC of Pseudomonas putida S16 were distinctly higher when grown in nicotine medium than in glycerol medium.

No MeSH data available.


Related in: MedlinePlus