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Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

Ding W, Si M, Zhang W, Zhang Y, Chen C, Zhang L, Lu Z, Chen S, Shen X - Sci Rep (2015)

Bottom Line: Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis.Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum.Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China.

ABSTRACT
Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51 kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2 kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30°C, and interestingly, it could utilize NAD(+) and NADP(+) as coenzymes with similar efficiency and showed no obvious difference toward NAD(+) and NADP(+). In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of vdh and the complemented strain vdh(pXMJ19-vdh) grown on mineral salts medium containing 8mM vanillin (A), 5mM 3-hydroxybenzaldehyde (B), 8mM p-hydroxy benzaldehyde (C), 5mM 3,4-dihydroxybenzaldehyde (D), 3mM ferulic acid (E) and caffeic acid (F), respectively. WT(pXMJ19), vdh(pXMJ19), vdh(pXMJ19-vdh).
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f2: Phenotypic characterization of vdh and the complemented strain vdh(pXMJ19-vdh) grown on mineral salts medium containing 8mM vanillin (A), 5mM 3-hydroxybenzaldehyde (B), 8mM p-hydroxy benzaldehyde (C), 5mM 3,4-dihydroxybenzaldehyde (D), 3mM ferulic acid (E) and caffeic acid (F), respectively. WT(pXMJ19), vdh(pXMJ19), vdh(pXMJ19-vdh).

Mentions: To further characterize the vanillin dehydrogenase activity of VDHATCC13032, a mutant strain (vdhATCC13032) was obtained by homologous recombination based gene knock-out. Growth analyses of wild type strain and vdhATCC13032 were conducted in liquid media at 30C, using different substrates such as vanillin (8mM), p-hydroxybenzaldehyde (8mM), 3,4-dihydroxybenzaldehyde (5mM), 3-hydroxybenzaldehyde (5mM), ferulic acid (3mM), caffeic acid (3mM), p-cresol (5mM), cinnamyl aldehyde (5mM) and syringaldehyde (2.5mM)) as the sole carbon and energy source. Compared to the wild type, the vdhATCC13032 mutant showed remarkably reduced ability to grow with the above mentioned aromatic compounds (Fig. 2). When complemented with plasmid pXMJ19-vdhATCC13032, the growth ability of the mutant strain could be restored close to that of the wild type (Fig. 2). However, the wild type, the vdhATCC13032 mutant and the complementary strain showed no difference when grown in p-cresol, cinnamyl aldehyde and syringaldehyde (data not shown).


Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

Ding W, Si M, Zhang W, Zhang Y, Chen C, Zhang L, Lu Z, Chen S, Shen X - Sci Rep (2015)

Phenotypic characterization of vdh and the complemented strain vdh(pXMJ19-vdh) grown on mineral salts medium containing 8mM vanillin (A), 5mM 3-hydroxybenzaldehyde (B), 8mM p-hydroxy benzaldehyde (C), 5mM 3,4-dihydroxybenzaldehyde (D), 3mM ferulic acid (E) and caffeic acid (F), respectively. WT(pXMJ19), vdh(pXMJ19), vdh(pXMJ19-vdh).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306973&req=5

f2: Phenotypic characterization of vdh and the complemented strain vdh(pXMJ19-vdh) grown on mineral salts medium containing 8mM vanillin (A), 5mM 3-hydroxybenzaldehyde (B), 8mM p-hydroxy benzaldehyde (C), 5mM 3,4-dihydroxybenzaldehyde (D), 3mM ferulic acid (E) and caffeic acid (F), respectively. WT(pXMJ19), vdh(pXMJ19), vdh(pXMJ19-vdh).
Mentions: To further characterize the vanillin dehydrogenase activity of VDHATCC13032, a mutant strain (vdhATCC13032) was obtained by homologous recombination based gene knock-out. Growth analyses of wild type strain and vdhATCC13032 were conducted in liquid media at 30C, using different substrates such as vanillin (8mM), p-hydroxybenzaldehyde (8mM), 3,4-dihydroxybenzaldehyde (5mM), 3-hydroxybenzaldehyde (5mM), ferulic acid (3mM), caffeic acid (3mM), p-cresol (5mM), cinnamyl aldehyde (5mM) and syringaldehyde (2.5mM)) as the sole carbon and energy source. Compared to the wild type, the vdhATCC13032 mutant showed remarkably reduced ability to grow with the above mentioned aromatic compounds (Fig. 2). When complemented with plasmid pXMJ19-vdhATCC13032, the growth ability of the mutant strain could be restored close to that of the wild type (Fig. 2). However, the wild type, the vdhATCC13032 mutant and the complementary strain showed no difference when grown in p-cresol, cinnamyl aldehyde and syringaldehyde (data not shown).

Bottom Line: Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis.Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum.Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, PR China.

ABSTRACT
Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51 kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2 kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30°C, and interestingly, it could utilize NAD(+) and NADP(+) as coenzymes with similar efficiency and showed no obvious difference toward NAD(+) and NADP(+). In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

No MeSH data available.


Related in: MedlinePlus