TY - JOUR
T1 - Probing the role of the conserved Arg174 in formate dehydrogenase by chemical modification and site-directed mutagenesis
AU - Alqarni, Mohammed Hamed
AU - Foudah, Ahmed Ibrahim
AU - Mohamed Muharram, Magdy
AU - Budurian, Haritium
AU - Labrou, Nikolaos E.
N1 - Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/3/1
Y1 - 2021/3/1
N2 - 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 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.
AB - 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 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.
KW - Formate dehydrogenase
KW - NAD+ binding site
KW - Site-directed mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=85102698332&partnerID=8YFLogxK
U2 - 10.3390/molecules26051222
DO - 10.3390/molecules26051222
M3 - Article
C2 - 33668802
AN - SCOPUS:85102698332
SN - 1420-3049
VL - 26
JO - Molecules
JF - Molecules
IS - 5
M1 - 1222
ER -