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.
Highlights
Microbial degradation of N-heterocyclic compounds, including xanthine, quinoline, nicotinate, and nicotine, frequently requires molybdenum hydroxylases
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] clusters[3]
In consideration of the conserved biosynthetic pathway of molybdenum cofactor in all bacteria, gene annotation was quite credible. These predicted genes involved in molybdate uptake, molybdopterin biosynthesis and molybdenum cofactor further modification. Four of these genes (PPS_1556, encoding for molybdenum cofactor biosynthesis protein A; PPS_2936, encoding for molybdopterin-guanine dinucleotide biosynthesis protein A; PPS_4063, encoding for molybdenum cofactor biosynthesis protein; PPS_4397, encoding for molybdopterin biosynthesis molybdopterin synthase large subunit (MoaE) protein) were successfully knocked out by using the suicide plasmid pK18mob, which were confirmed by PCR analysis (Fig. 2B)
Summary
Microbial degradation of N-heterocyclic compounds, including xanthine, quinoline, nicotinate, and nicotine, frequently requires molybdenum hydroxylases. 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. Nicotine is well known to be harmful to human health and can cross biological membranes and blood-brain barrier [2] Microorganisms such as Pseudomonas are important for degrading nicotine, and the pyrrolidine pathway of nicotine degradation has been systematically unraveled in Pseudomonas putida strain S163–6. 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] clusters[3].
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