Limits...
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.


Biotransformation of vanillin (A) and 3,4-dihydroxybenzaldehyde (B) with purified VDHATCC13032.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4306973&req=5

f5: Biotransformation of vanillin (A) and 3,4-dihydroxybenzaldehyde (B) with purified VDHATCC13032.

Mentions: The catalytic activity of VDHATCC13032 was further confirmed by LC-MS analysis using the selected substrates. Purified VDHATCC13032 enabled the conversion of vanillin to vanillate (as shown in Figure 5). The HPLC results indicated that the production of vanillate in the presence of NAD+ and VDHATCC13032 was coupled with the decrease of the vanillin concentration (Fig. 5A). The production of vanillate was also validated by MS analysis (Data not shown). Purified VDHATCC13032 also considerably converted 3,4-dihydroxybenzaldehyde to 3,4-dihydroxybenzoic acid (Fig. 5B).


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)

Biotransformation of vanillin (A) and 3,4-dihydroxybenzaldehyde (B) with purified VDHATCC13032.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Biotransformation of vanillin (A) and 3,4-dihydroxybenzaldehyde (B) with purified VDHATCC13032.
Mentions: The catalytic activity of VDHATCC13032 was further confirmed by LC-MS analysis using the selected substrates. Purified VDHATCC13032 enabled the conversion of vanillin to vanillate (as shown in Figure 5). The HPLC results indicated that the production of vanillate in the presence of NAD+ and VDHATCC13032 was coupled with the decrease of the vanillin concentration (Fig. 5A). The production of vanillate was also validated by MS analysis (Data not shown). Purified VDHATCC13032 also considerably converted 3,4-dihydroxybenzaldehyde to 3,4-dihydroxybenzoic acid (Fig. 5B).

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.