Functional Characterization Of A Vanillin Dehydrogenase In Corynebacterium Glutamicum

Vanillin is type of aromatic compounds widely distributed in natural environments and is harmful to the environments and ecology. Recent years’ focusd on vanillin dehydrogenase(VDH) is confined to Gram negative bacteria, especially the Pseudomonas. In contrast, studies on VDH in Gram positive bacteria remain still limited. Previous reports suggested that the type and function of VDH in Gram positive bacteria differ from these in Gram negative bacteria. Thus, to further study the function of VDH in Gram positive bacteria is of great interest. Corynebacterium glutamicum can grow using various aromatic compounds including vanillin as the sole carbon source. Based on sequence alignment and homology analysis, the gene ncgl2578 was predicted to encode the VDH in Corynebacterium glutamicum. However, characterization of this gene has not been conducted.In the present study, we firstly constructed a phylogenetic tree including the VDH in Corynebacterium glutamicum and reported VDH homologs from other species. The results suggested that VDH in Corynebacterium glutamicum formed an independent clade among the family of this enzyme. Thus, further study on the function of this gene contributes to our understanding of the functional diversity and character of this enzyme family. We subsequently constructed the mutants of the VDH, and characterized the effects of vdh deletion in Corynebacterium glutamicum on aromatic compounds utilization. The p-hydroxybenzaldehyde, 3, 4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde were used as substrates for investigation and the wild-type strain, vdh deletion strain and the complementary strain were compared. The results indicated that the deletion caused a defective growth of the Corynebacterium glutamicum when vanillin, 3, 4-dihydroxybenzaldehyde or p-hydroxybenzaldehyde was used as sole carbon source. However, the growth state of the vdh gene complementary strain was quite similar as the wild-type strain. These results demonstrated that the VDH is essential for the degradation of vanillin, 3,4-dihydroxybenzaldehyde or p-hydroxybenzaldehyde.To further study the function of VDH, we expressed and purified this protein in vitro. Using HPLC, we then tested its role in transformation of vanillin and 3, 4-dihydroxybenzaldehyde to vanillic acid and 3,4-Dihydroxybenzoic acid respectively. Moreover, we characterized the molecular weight, the aggregation morphology, the optimum temperature, the optimum pH, the scope of substrates, the Km value and the active residues of the VDH. Through the SDS-PAGE detection, the molecular weight of this protein is about 50 kDa. The native PAGE detected the existence of tetramer, trimer and dimer when the VDH was in its active state. Based on the catalytic analysis, the optimum temperature for the VDH was 30℃ whereas the optimum pH was pH 7.0. Moreover, the protein could utilize both NAD+ and NADP+ as the cofactor for catalytic reaction. The VDH could in vitro catalyze reactions of a wide range of aromatic compounds, including vanillin, 3,4-dihydroxybenzaldehyde and p-hydroxybenzaldehyde, benzalacet aldehyde, syringaldehyde, and phenyl acetaldehyde. Therefore, the VDH encoded by ncgl2578 in the Corynebacterium glutamicum is an enzyme with a broad range of substrates, multiply functional residues and is necessary for utilization of vanillin and similar compounds.In addition, through sequence alignment and comparison with previously reported VDHs, we further predicted the conserved functional regions of the VDH in Corynebacterium glutamicum. The prediction was confirmed by the site mutation and enzymatic assay. The results demonstrated the different roles of the conserved sites: all of the five conserved sites were indispensable in the interaction between VDHATCC13032 and NAD+; mutation of E199 did not affected the binding of VDHATCC13032 to NADP+; K180,E258,and C292 played important roles in mediating the binding of the VDHATCC13032 protein to substrates.In summary, the present study contributes to further understanding of the VDH diversity and the mechanisms underlying the aromatic compounds metabolism in Corynebacterium glutamicum.