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Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis
| Content Provider | MDPI |
|---|---|
| Author | Alqarni, Mohammed Foudah, Ahmed Muharram, Magdy Budurian, Haritium Labrou, Nikolaos |
| Copyright Year | 2021 |
| Description | The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD+. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (KD = 0.66 ± 0.15 mM, first order maximum rate constant k3 = 0.198 ± 0.06 min−1). NAD+ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD+ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH. |
| Starting Page | 1222 |
| e-ISSN | 14203049 |
| DOI | 10.3390/molecules26051222 |
| Journal | Molecules |
| Issue Number | 5 |
| Volume Number | 26 |
| Language | English |
| Publisher | MDPI |
| Publisher Date | 2021-02-25 |
| Access Restriction | Open |
| Subject Keyword | Molecules Biochemistry and Molecular Biology Formate Dehydrogenase Nad+ Binding Site Site-directed Mutagenesis |
| Content Type | Text |
| Resource Type | Article |
