Met-Leu

NAD+-dependent formate dehydrogenases (FDHs) are extensively used in the regeneration of NAD(P)H and the reduction of CO2 to formate. In addition to their industrial importance, FDHs also play a crucial role in the maintenance of a reducing environment to combat oxidative stress in plants. Therefore, it is important to investigate the response of NAD+-dependent FDH against both temperature and H2O2, to understand the defense mechanisms, and to increase its stability under oxidative stress conditions. In the present study, we characterized the oxidative and thermal stability of NAD+-dependent FDH isolated from cotton, Gossypium hirsutum (GhFDH), by investigating the effect of Met/Leu substitutions in the positions of 225, 234, and 243. Results showed that the single mutant, M234L (0.72 s-1 mM-1), and the triple mutant, M225L/M234L/M243L (0.55 s-1 mM-1), have higher catalytic efficiency than the native enzyme. Substitution of methionine by leucine on the position of 243 increased the free energy gain by 670 J mol-1. The most remarkable results in chemical stability were seen for double and triple mutants, cumulatively. Double and triple substitution of Met to Leu (M225L/M243L and M225L/M243L/M234L) reduce the kefin by a factor of 2 (12.3×10-5 and 12.8×10-5 s-1, respectively.Key points· The closer the residue to NAD+, in which we substituted methionine to leucine, the lower the stability against H2O2 we observed.· The significant gain in the Tm value for the M243L mutant was observed as +5°C.· Residue 234 occupies a critical position for oxidation defense mechanisms. Graphical abstract (a) Methionine amino acids on the protein surface are susceptible to oxidative stress and can be converted to methionine sulfoxide by reactive oxygen derivatives (such as hydrogen peroxide). Therefore, they are critical regions in the change of protein conformation and loss of activity. (b) Replacing the amino acid methionine, which is susceptible to oxidation due to the sulfur group, with the oxidation-resistant leucine amino acid is an important strategy in increasing oxidative stability.

Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes. The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM. All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli. All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium. Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE. The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM. The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE. Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure. Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations.

Neutrophils constitute the first line of defence against bacterial invasion. They migrate to infected tissues along a concentration gradient of chemoattractant molecules, the most important of which is for-Met-Leu-Phe-OH (fMLP). Different responses arise from formylpeptides binding to different isoforms of the specific receptor. The aim of the studies reported herein was to clarify (i) the role of fMLP-OMe amide bonds in receptor-ligand cross-linking, (ii) the nature of the group occupying the N- and C-terminal positions, (iii) the features peculiar to the Met, Leu, and Phe receptor pockets, and (iv) the features which determine the specific neutrophil response.