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

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 (K_D = 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.

Keywords: NAD+ binding site; formate dehydrogenase; site-directed mutagenesis.

Figure 1

The structure of the the reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB) (A). The structure of the ADP part of NAD⁺ is also presented (B), for comparison. Where R: nicotinamide ribose part of NAD⁺.

Figure 2

Structural analysis. (A) Aminoacid sequence alignments of NAD⁺-binding region of plant, bacteria, fungi and yeasts FDHs. Abbreviations and National Center for Biotechnology Information (NCBI) accession codes are: FDH_Cb, CAA09466; FDH_Sc, NP_015033, FDH_Pan, P33677; FDH_Cal, XP_711169; FDH_Psp, P33160; FDH_Tsp, BAC92737; FDH_Ath, NP_196982; FDH_Stu, Q07511; FDH_Osa, NP_001057666; FDH_Qro, CAE12168; FDH_Hvu, Q9ZRI8; FDH_Aca, AAV67968; FDH_Ani, XP_664129; FDH_Mgr, AAW69358; FDH_Gze, XP_386303; FDH_Ncr, XP_961202. The alignments were created using Clustal O. The secondary structure of CbFDH (PDB code 2FSS) and numbering are shown above the alignment. The arrow below the alignments depicts Arg174. (B) Asteroid Plot of interactions of Arg174 in NAD⁺-free (i) (Protein Data Bank code: 5dna) and NAD⁺-bound (ii) (Protein Data Bank code: 5dn9) structures. The inner ring indicates first shell of immediate atomic contacts. The outer ring indicates second shell of extended atomic contacts. The size of the circle is proportional to the total number of contacts that each residue is involved in with any of the residues in the ring inward to it. The diagrams were created by Protein Contact Atlas [38]. (C) Diagram of the subunit of CbFDH with NAD⁺ bound to the active site. (D) Movement of Arg174 upon NAD binding. (i) The conformation of Arg174 in NAD⁺-bound structure (PDB code: 5dn9, colored grew). (ii) The conformation of Arg174 in NAD⁺-free structure (PDB code: 5dna, colored brown). Both structures (5dn9 and 5dna) were superimposed. (E) Coulombic surface coloring of the electrostatic potential in the NAD⁺ binding site. NAD⁺ is shown in stick representation and colored according to the atom type.

Figure 3

Inactivation of recombinant CbFDH by AMPS-HDB. Enzyme was incubated in the absence (♦) and in the presence of AMPS-HDB: 0.05 mM (•); 0.1 mM (■); 0.2 mM (▲); 0.3 mM (▼); 0.5 mM (◊); at pH 8.0 and 25 °C. At the times indicated, aliquots were withdrawn and assayed for enzymatic activity.

Figure 4

Dependence of the pseudo-first-order rate constant of inactivation (k_obs) on the concentration of AMPS-HDB. CbFDH was incubated with various concentrations of AMPS-HDB (0–0.5 mM) and the pseudo-first-order rate constants for the inactivation reaction were calculated from the plots as illustrated in Figure 1.

Scheme 1

The reaction of an Arg residue with dicarbonyl compound in borate buffer.

Figure 5

The effects of nucleotides and mutation at position 174 on the inactivation of CbFDH by AMPS-HDB. (A) Time course of inactivation of CbFDH by AMPS-HDB (0.1 mΜ) at pH 8.0 and 25 °C, in the absence (●) or in the presence of NAD⁺ (■, 1 mM) or NADH (▲, 1 mM). (B) Inactivation of the mutant enzyme Arg174Asn by AMPS-HDB (0.1 mΜ) (■). At the times indicated, aliquots were withdrawn and assayed for enzymatic activity.

Figure 6

Effect of temperature on V_max for the wild-type and the mutant Arg174Asn enzyme. The data were fitted to the Arrhenius equation for the wild-type, (●); and for the mutant (○) enzyme.

Figure 7

Effect of point mutation on thermal stability. (A). The residual activities of the wild-type enzyme (●) and the mutant enzyme Arg174Asn (■) were measured after heat treatment at various temperatures (°C). (B). Time course of thermal inactivation of the wild-type enzyme (●) and the mutant enzyme Arg174Asn (■) at 60 °C. At indicated times, enzyme samples were removed and assayed for residual FDH activity.