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

Illava, Gabrielle; Gillilan, Richard E.; Ando, Nozomi

doi:10.1101/2023.05.18.541370

Cited by 1 publication

(1 citation statement)

References 52 publications

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“…S3 C , Inset ) exposure was an indication that the 6-coordinate, His-on CH 3 -Cob(III) state did not undergo photolysis or photoreduction. Finally, a fully anoxic setup ( 34 ) was required for the highly oxygen-sensitive Cob(I) MetH ( 16 , 32 ), where the sample loading, pumps, and waste lines were contained in an in-line anoxic chamber at the beamline ( SI Appendix , SI Methods ). The oxidation state was verified by the characteristic, sharp peak at ~390 nm ( 28 , 32 ) observed both before ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Watkins

Wang

Burnim

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH 3 -H 4 folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S -adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multidomain 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 investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length 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 CH 3 -H 4 folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, 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.

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