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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.


Rooted neihbor-joining tree constructed from the amino acid sequence of 14 vanillin dehydrogenases.Boot strap confidence limits (expressed as percentage) are shown at nodes. Multiple-sequence alignment was done by using ClustalX 1.83 based on the amino acid sequences of the following enzymes: putative VDH of C. glutamicum ATCC13032 (NP_601867.1), benzaldehyde dehydrogenase of Rhodococcus erythropolis PR43839, VDH of Rhodococcus jostii RHA140, VDH of Sphingomonas paucimobilis SYK-61314, benzaldehyde dehydrogenase of Pseudomonas putida CSV861141, VDH of Pseudomonas putida WCS35842, VDH of Pseudomonas putida KT24401043, VDH of Pseudomonas sp. HR19924, VDH of Amycolatopsis sp. ATCC 3911621, VDH of Pseudomonas fluorescens3244, aldehyde dehydrogenase (NAD+) of Pseudonocardia sp. P1(ZP_08121488.1), benzaldehyde dehydrogenase (NAD+) of Saccharopolyspora erythraea NRRL 2338(YP_001105347.1), benzaldehyde dehydrogenase (NAD+) of Burkholderia cenocepacia AU 1054(YP_621190.1), and aldehyde dehydrogenase of Amycolatopsis mediterranei U32 (AMED_0881). Calculations were performed by using the neighbor-joining method. Enzymes with verified vanillin dehydrogenase activity are shown in boldface type.
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f1: Rooted neihbor-joining tree constructed from the amino acid sequence of 14 vanillin dehydrogenases.Boot strap confidence limits (expressed as percentage) are shown at nodes. Multiple-sequence alignment was done by using ClustalX 1.83 based on the amino acid sequences of the following enzymes: putative VDH of C. glutamicum ATCC13032 (NP_601867.1), benzaldehyde dehydrogenase of Rhodococcus erythropolis PR43839, VDH of Rhodococcus jostii RHA140, VDH of Sphingomonas paucimobilis SYK-61314, benzaldehyde dehydrogenase of Pseudomonas putida CSV861141, VDH of Pseudomonas putida WCS35842, VDH of Pseudomonas putida KT24401043, VDH of Pseudomonas sp. HR19924, VDH of Amycolatopsis sp. ATCC 3911621, VDH of Pseudomonas fluorescens3244, aldehyde dehydrogenase (NAD+) of Pseudonocardia sp. P1(ZP_08121488.1), benzaldehyde dehydrogenase (NAD+) of Saccharopolyspora erythraea NRRL 2338(YP_001105347.1), benzaldehyde dehydrogenase (NAD+) of Burkholderia cenocepacia AU 1054(YP_621190.1), and aldehyde dehydrogenase of Amycolatopsis mediterranei U32 (AMED_0881). Calculations were performed by using the neighbor-joining method. Enzymes with verified vanillin dehydrogenase activity are shown in boldface type.

Mentions: Based on BLAST Search and genome sequence analysis, the gene coding for a putative vanillin dehydrogenase (ncgl2578, named as vdhATCC13032 in this study) was identified, which composed of 1,455bp and encoded a protein of 484 amino acids with a theoretical molecular mass of 51.5kDa. The vdhATCC13032 shares 35%, 41% and 59% amino acid sequence identity with the vdh genes from Pseudomonas aeruginosa DK2, Rhodococcus jostii RHA1 and Pseudomonas fluorescens, respectively. To further assess the phylogenetic relationship between vdhATCC13032 and vdh genes from other bacteria, a multiple-sequence alignment was conducted using ClustalX 1.83 (Fig. 1). The results showed that the vdhATCC13032 from C. glutamicum forms an independent cluster on the phylogenetic tree and exhibits clear evolutionary distance with already verified vdh genes from other bacteria. These results suggested that vdh from C. glutamicum may therefore represent a new vanillin dehydrogenase branch and the vdhATCC13032 represents the first vanillin dehydrogenase characterized in detail within this gene family.


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)

Rooted neihbor-joining tree constructed from the amino acid sequence of 14 vanillin dehydrogenases.Boot strap confidence limits (expressed as percentage) are shown at nodes. Multiple-sequence alignment was done by using ClustalX 1.83 based on the amino acid sequences of the following enzymes: putative VDH of C. glutamicum ATCC13032 (NP_601867.1), benzaldehyde dehydrogenase of Rhodococcus erythropolis PR43839, VDH of Rhodococcus jostii RHA140, VDH of Sphingomonas paucimobilis SYK-61314, benzaldehyde dehydrogenase of Pseudomonas putida CSV861141, VDH of Pseudomonas putida WCS35842, VDH of Pseudomonas putida KT24401043, VDH of Pseudomonas sp. HR19924, VDH of Amycolatopsis sp. ATCC 3911621, VDH of Pseudomonas fluorescens3244, aldehyde dehydrogenase (NAD+) of Pseudonocardia sp. P1(ZP_08121488.1), benzaldehyde dehydrogenase (NAD+) of Saccharopolyspora erythraea NRRL 2338(YP_001105347.1), benzaldehyde dehydrogenase (NAD+) of Burkholderia cenocepacia AU 1054(YP_621190.1), and aldehyde dehydrogenase of Amycolatopsis mediterranei U32 (AMED_0881). Calculations were performed by using the neighbor-joining method. Enzymes with verified vanillin dehydrogenase activity are shown in boldface type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Rooted neihbor-joining tree constructed from the amino acid sequence of 14 vanillin dehydrogenases.Boot strap confidence limits (expressed as percentage) are shown at nodes. Multiple-sequence alignment was done by using ClustalX 1.83 based on the amino acid sequences of the following enzymes: putative VDH of C. glutamicum ATCC13032 (NP_601867.1), benzaldehyde dehydrogenase of Rhodococcus erythropolis PR43839, VDH of Rhodococcus jostii RHA140, VDH of Sphingomonas paucimobilis SYK-61314, benzaldehyde dehydrogenase of Pseudomonas putida CSV861141, VDH of Pseudomonas putida WCS35842, VDH of Pseudomonas putida KT24401043, VDH of Pseudomonas sp. HR19924, VDH of Amycolatopsis sp. ATCC 3911621, VDH of Pseudomonas fluorescens3244, aldehyde dehydrogenase (NAD+) of Pseudonocardia sp. P1(ZP_08121488.1), benzaldehyde dehydrogenase (NAD+) of Saccharopolyspora erythraea NRRL 2338(YP_001105347.1), benzaldehyde dehydrogenase (NAD+) of Burkholderia cenocepacia AU 1054(YP_621190.1), and aldehyde dehydrogenase of Amycolatopsis mediterranei U32 (AMED_0881). Calculations were performed by using the neighbor-joining method. Enzymes with verified vanillin dehydrogenase activity are shown in boldface type.
Mentions: Based on BLAST Search and genome sequence analysis, the gene coding for a putative vanillin dehydrogenase (ncgl2578, named as vdhATCC13032 in this study) was identified, which composed of 1,455bp and encoded a protein of 484 amino acids with a theoretical molecular mass of 51.5kDa. The vdhATCC13032 shares 35%, 41% and 59% amino acid sequence identity with the vdh genes from Pseudomonas aeruginosa DK2, Rhodococcus jostii RHA1 and Pseudomonas fluorescens, respectively. To further assess the phylogenetic relationship between vdhATCC13032 and vdh genes from other bacteria, a multiple-sequence alignment was conducted using ClustalX 1.83 (Fig. 1). The results showed that the vdhATCC13032 from C. glutamicum forms an independent cluster on the phylogenetic tree and exhibits clear evolutionary distance with already verified vdh genes from other bacteria. These results suggested that vdh from C. glutamicum may therefore represent a new vanillin dehydrogenase branch and the vdhATCC13032 represents the first vanillin dehydrogenase characterized in detail within this gene family.

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.