Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

Abstract

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

Graphical Abstract

Substrate binding residues and Hg²⁺ sensitive residue in central domain of JL-GDH

Key words:

• very large glutamate dehydrogenase
• Janthinobacterium lividum
• glutamate-binding residues
• inhibition by Hg²⁺
• activation by Hg²⁺

Acknowledgments

We are grateful to Masaki Oyama and Shota Shiraishi for their excellent technical assistance. We would like to thank Enago (www.enago.jp) for the English language review.