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

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1222
Author(s):  
Mohammed Hamed Alqarni ◽  
Ahmed Ibrahim Foudah ◽  
Magdy Mohamed Muharram ◽  
Haritium Budurian ◽  
Nikolaos E. Labrou
Keyword(s):  
Formate Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Inactivation Kinetics ◽  
Candida Boidinii ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Complete Inactivation ◽  
Modelling Studies

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.

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

scholarly journals Site-directed mutagenesis of the formate dehydrogenase active centre: role of the His332-Gln313pair in enzyme catalysis

FEBS Letters ◽  
1996 ◽  
Vol 390 (1) ◽  
pp. 104-108 ◽  
Author(s):  
Vladimir I. Tishkov ◽  
Andrey D. Matorin ◽  
Alexandra M. Rojkova ◽  
Vladimir V. Fedorchuk ◽  
Pavel A. Savitsky ◽  
...  
Keyword(s):  
Enzyme Catalysis ◽  
Formate Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Centre

scholarly journals Function of the 90-loop (Thr90–Glu100) region of staphylokinase in plasminogen activation probed through site-directed mutagenesis and loop deletion

2002 ◽  
Vol 365 (2) ◽  
pp. 379-389 ◽  
Author(s):  
Govindan RAJAMOHAN ◽  
Monika DAHIYA ◽  
Shekhar C. MANDE ◽  
Kanak L. DIKSHIT
Keyword(s):  
Active Site ◽  
Complex Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Kringle Domains ◽  
Lysine Residues ◽  
Modelling Studies ◽  
Activator Complex

Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr90—Glu100) for the docking of the substrate PG to the SAK—Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr92—Glu100) from the SAK 90-loop, resulted in ≈60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKΔ90) and Pm. The preformed activator complex of SAKΔ90 with Pm, however, displayed a 50–60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK—Pm complex displayed ∼50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKΔ90. Lysine residues (Lys94, Lys96, Lys97 and Lys98) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAKK97A and SAKK98A exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the Km values of PG towards the activator complex of these two SAK mutants were 4–6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys97 and Lys98 residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK—Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.

Sign in / Sign up

Export Citation Format

Share Document