Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy

Abstract

Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH3-H4folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling ofS-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies onEscherichia coliMetH have shown that this flexible, multi-domain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a "divide and conquer" approach. In this study, we investigateE. coliMetH and a thermophilic homolog fromThermus filiformisusing small-angle X-ray scattering (SAXS), single-particle cryo-electron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present the first structural description of MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH3-H4folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of theT. filiformisMetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator

“…The effectiveness of batchmode anSAXS was recently demonstrated by an investigation of the cobalamin-dependent methionine synthase, in which the highly oxygen-sensitive Cob(I) oxidation state of the enzyme was maintained throughout data collection. 39 In this current study, we report the first demonstration of SEC-anSAXS through analysis of the oxygen-dependent oligomerization and DNA binding of E. coli FNR. Because E. coli FNR can exist as a mixture of oligomeric species, the introduction of in-line chromatography was an essential development.…”

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator

“…Here, we introduced an in-line system at CHESS beamline ID7A that enables both chromatography-coupled SAXS and batch-mode SAXS under anoxic conditions. The effectiveness of batch-mode anSAXS was recently demonstrated by an investigation of the cobalamin-dependent methionine synthase, in which the highly oxygen-sensitive Cob(I) oxidation state of the enzyme was maintained throughout data collection ( 40 ). In this current study, we report the first demonstration of SEC-anSAXS through analysis of the oxygen-dependent oligomerization and DNA binding of E. coli FNR.…”

Architecture of the human G-protein-methylmalonyl-CoA mutase nanoassembly for B12delivery and repair

“…Ligand-triggered conformational changes have been reported in bacterial methionine synthase, although the mechanism of cofactor loading awaits elucidation (42). In Aquincola tertiaricarbonis, a MeaB like gene is in the same operon as the large subunit of cobalamin-dependent hydroxyisobutyryl-CoA mutase (43).…”

“…The dramatic stacked to unstacked conformational change could be a common strategy used by B 12 -dependent enzymes, particularly those that bind the cofactor in the base-off conformation. Ligand-triggered conformational changes have been reported in bacterial methionine synthase, although the mechanism of cofactor loading awaits elucidation (42). In Aquincola tertiaricarbonis , a MeaB like gene is in the same operon as the large subunit of cobalamin-dependent hydroxyisobutyryl-CoA mutase (43).…”