Metal active site elasticity linked to activation of homocysteine in methionine synthases

Abstract

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.cobalamin-dependent methionine synthase ͉ cobalamin-independent methionine synthase ͉ methyl transferases ͉ zinc enzymes ͉ zinc inversion

Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

“…This Zn-binding active site Cys is buried in the MetE structures of Thermotoga maritima and Streptococcus mutans (PDB code 2NQ5) (Supplementary Fig. S7), but the elastic and flexible nature of this catalytic Zn center has been demonstrated (28). However, in contrast to E. coli and B. subtilis, NaOCl stress did not cause methionine starvation since growth was not resumed by addition of Met to the growth medium after 30 and 60 min of NaOCl treatment (Supplementary Fig.…”

S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics

“…The reaction of GSH with hypochloric acid is spontaneous and does not involve conjugating enzymes such as glutathione S-transferases (GSTs) (83 (84,85). MetE is S-bacillithiolated at the non-essential Cys719 and the Zn-binding ligand Cys730 and the methionine synthase paralog YxjG is S-bacillithiolated at Cys346 that aligns with Cys730 in MetE.…”

Characterization of selenocysteine methyltransferases from Astragalus species with contrasting selenium accumulation capacity

“…The Ala to Thr-184 substitution may have modulated the structure of the catalytic site of the enzyme, allowing it to increase its turnover rate without altering the substrate binding affinity. The equivalent Thr-147 residue of the Tm-MetH protein donates a hydrogen bond to the Zn-bound thiol group of HoCys to facilitate the methylation reaction (Koutmos et al, 2008) and we can speculate that this residue may interact in a similar manner with the seleno group of SeCys. The Thr residue side chain contains a b-carbon hydroxyl group that can efficiently coordinate a hydrogen bond.…”

“…Crystallographic analysis of the Tm-MetH structure revealed a (ba) 8 barrel with a catalytic zinc-binding site, near the top of the barrel, that coordinates binding of HoCys and is assembled from Cys-207, Cys-272 and Cys-273 residues (Evans et al, 2004;Koutmos et al, 2008). Importantly, this ternary geometrical arrangement of the catalytic site positions the sulphur group of HoCys in close proximity, to Thr-147, which is responsible for donating a hydrogen bond and facilitating the methyl transfer reaction.…”

“…To determine whether polymorphisms within the SMT gene could account for the observed differences in SMT activity (Millian and Garrow, 1998;Peariso et al, 1998;Koutmos et al, 2008). Only 13 amino acid differences occur between AbSMT and AdSMT, including eight non-conservative amino acid changes and a deletion.…”