Mechanism of the 6-Hydroxy-3-succinoyl-pyridine 3-Monooxygenase Flavoprotein from Pseudomonas putida S16

Yu, Hao; Hausinger, Robert P.; Tang, Hong Zhi; Xu, Ping

doi:10.1074/jbc.m114.558049

Cited by 28 publications

(32 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fe-S clusters can be identified from the characteristic absorption peak of purified VppA, and His-tag-specific staining indicated the existence of a [2Fe-2S]-binding subunit. However, no other extra bands were observed by SDS-PAGE, and the UV-visible spectrum did not show the characteristic peak of a flavin cofactor (10). These results suggested that VppA does not have an FAD-binding subunit.…”

Section: Discussionmentioning

confidence: 63%

“…The pyridine hydroxylases can be subclassified as ␣-, ␤-, or ␥-position hydroxylases according to the hydroxylation position on the pyridine ring (10). VppA is classified as a pyridine ring ␣-position hydroxylase based on its catalytic reaction.…”

Section: Discussionmentioning

confidence: 99%

“…1) (6)(7)(8)(9). The molecular mechanisms of nicotine degradation by the pyridine pathway and the pyrrolidine pathway have been elucidated in detail by the research groups of Brandsch (3) and Xu et al (2,10,11), respectively. However, the genes involved in several catalyzing steps of the VPP pathway are still unknown.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Tang

et al. 2015

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

bOchrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppA S and vppA L [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5␣ and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FADbinding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase. N icotine is the main toxic compound in tobacco, and it accumulates with tobacco wastes during the processing of tobacco products. Nicotine can easily spread into the environment due to its water solubility and endanger the health of humans and other organisms (1). Microbial biodegradation is one of the best remediation strategies to remove nicotine from the environment (2-4). A number of bacteria and fungi which use nicotine as a sole source of carbon, nitrogen, and energy for their growth have been isolated and identified (3, 4). Three nicotine degradation pathways have been proposed in bacteria based on the identification of intermediates: the pyridine pathway (3), the pyrrolidine pathway (2, 5), and the variant of pyridine and pyrrolidine pathways (the VPP pathway) (Fig. 1) (6-9). The molecular mechanisms of nicotine degradation by the pyridine pathway and the pyrrolidine pathway have been elucidated in detail by the research groups of Brandsch (3) and Xu et al. (2, 10, 11), respectively. However, the genes involved in several catalyzing steps of the VPP pathway are still unknown.Thus far, several bacteria that are able to degrade nicotine via the VPP pathway have been reported, including Ochrobactrum sp. strain SJY1 (6, 7), Agrobacterium tumefaciens S33 (12), Shinella sp. strain HZN7 (8), and a Pusillimonas strain (13). In the VPP pathway, nicotine degradation is initiated by a hydroxylation reaction to form 6-hydroxynicotine (6HN), which is then converted to 6-hydroxy-N-methylmyosmine (6HMM) and 6-hydroxypseudooxynicotine (6HPON). Subsequently, 6HPON is oxidized to form 6-hydroxy-3-succinoylpyridine (HSP), which is catabolized to fumaric acid through 2,5-dihy...

show abstract

Section: Discussionmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Tang

et al. 2015

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The enzyme is among a few flavin‐dependent monooxygenases that utilize hydroxypyridine compounds as substrates. Other hydroxypyridine–utilizing flavoenzymes include 5PAO, 6‐hydroxy‐3‐succinoyl‐pyridine 3‐monooxygenases (HspB) (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/14/13/163.html) , 6‐hydroxynicotinate 3‐monooxygenase (6HN3M) (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/14/13/114.html) , 2,6‐dihydroxypyridine 3‐monooxygenase (DHPH) (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/14/13/10.html) and the recently reported 5‐hydroxypicolinic acid 2‐monooxygenase (5HP2M) . These enzyme were found in the degradation pathways of pyridine derivatives such as nicotine in soil bacteria .…”

Section: Introductionmentioning

confidence: 99%

Selectivity of substrate binding and ionization of 2‐methyl‐3‐hydroxypyridine‐5‐carboxylic acid oxygenase

2015

View full text Add to dashboard Cite

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located 5 A away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had K d values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H 2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the K d for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only~40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two-to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the interaction between Tyr223 and the substrate is important for ensuring hydroxylation. These results highlight how the active site of a flavoenAbbreviations

show abstract

“…The activity of HSP hydroxylase was determined using the method of Tang et al [23,30]. The standard reaction mixture, containing 10 mM FAD, 1 mM HSP, 0.5 mM NADH, and 20 mM Tris-HCl buffer (pH 8.5), was preincubated for 2 min and then the reaction was started by adding the purified enzyme.…”

Section: Enzymatic Activity Assaymentioning

confidence: 99%

Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine

et al. 2017

View full text Add to dashboard Cite

A novel nicotine hydroxylase was isolated from Pseudomonas sp. ZZ-5 (HSPH ZZ ). The sequence encoding the enzyme was 1206 nucleotides long, and encoded a protein of 401 amino acids. Recombinant HSPH ZZ was functionally overexpressed in Escherichia coli BL21-Codon Plus (DE3)-RIL cells and purified to homogeneity after Ni-NTA affinity chromatography. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that the enzyme could efficiently catalyze the conversion of 6-hydroxy-3-succinoylpyridine (HSP) into 2,5-dihydroxypyridine (2,5-DHP) and succinic acid in the presence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The kinetic constants (K m , k cat , and k cat /K m ) of HSPH ZZ toward HSP were 0.18 mM, 2.1 s −1 , and 11.7 s −1 mM −1 , respectively. The optimum temperature, pH, and optimum concentrations of substrate and enzyme for 2,5-DHP production were 30 • C, 8.5, 1.0 mM, and 1.0 µM, respectively. Under optimum conditions, 85.3 mg/L 2,5-DHP was produced in 40 min with a conversion of 74.9%. These results demonstrated that HSPH ZZ could be used for the enzymatic production of 2,5-DHP in biotechnology applications.

show abstract

Mechanism of the 6-Hydroxy-3-succinoyl-pyridine 3-Monooxygenase Flavoprotein from Pseudomonas putida S16

Cited by 28 publications

References 43 publications

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Selectivity of substrate binding and ionization of 2‐methyl‐3‐hydroxypyridine‐5‐carboxylic acid oxygenase

Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine

Contact Info

Product

Resources

About