Probing the Role of the Conserved Arg174 in Formate Dehydrogenase by Chemical Modification and Site-Directed Mutagenesis.

Mohammed Hamed Alqarni,Nikolaos E Labrou,Ahmed Ibrahim Foudah,Haritium Budurian,Magdy Mohamed Muharram

doi:10.3390/molecules26051222

Abstract

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.

Highlights

NAD+ -dependent formate dehydrogenase (FDH, EC 1.2.1.2) catalyzes the reversible conversion of formate anion to carbon dioxide and two electrons [1,2,3]
The reactive adenosine derivative adenosine 50 -O-[S-(4-hydroxy-2,3-dioxobutyl)]thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position such that it can structurally mimics the pyrophosphate region of NAD+
In all FDHs, the first amino acid residue after the second Gly residue is an arginine residue (Arg174 according to Candida boidinii formate dehydrogenase (CbFDH) numbering, Figure 2A)

Summary

Introduction

NAD+ -dependent formate dehydrogenase (FDH, EC 1.2.1.2) catalyzes the reversible conversion of formate anion to carbon dioxide and two electrons [1,2,3]. FDHs, based on their structural features and cofactor requirements are classified into two groups [4]: the NAD+ -independent and the NAD+ -dependent. NAD+ -independent FDHs contain at the active site oxygen-labile compounds (e.g., tungsten, molybdenum, iron-sulfur clusters, selenocysteine). They are sensitive enzymes and NAD+ -independent FDHs appear to have limited suitability in bicatalysis and other biotechnological applications. NAD+ -dependent FDHs are stable enzymes and have been exploited in several applications [5,6,7]. A wide range of FDHs have found successful applications in the development of efficient NAD(H) regeneration systems [6,8,9,10,11], as well as CO2 -reduction systems [7,12,13,14,15,16]

Methods

Results

Conclusion