ACERT Highlights: Recent Highlights

National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

Recent Highlights

Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe–4S] to a [4Fe–4S] Cluster in Dph1–Dph2

All radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1–Dph2, require at least one [4Fe–4S](Cys)3 cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe–4S](Cys)3 cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1–Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe–4S] cluster is easily degraded into a [3Fe–4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe–4S] cluster in Dph1–Dph2 to a functional [4Fe–4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.

Publication: J. Am. Chem. Soc. 143, 9314-9319 (2021); PMC8251694.

Figure: Dph3 activates aerobically purified Dph1–Dph2. (A) In vitro reconstitution of the first step of diphthamide biosynthesis with aerobically purified Dph1–Dph2. The reaction was carried out aerobically. The reaction time is labeled on the right. (B) In vitro reconstitution of the first step of diphthamide biosynthesis with anaerobically purified Dph1–Dph2. The reaction was carried out anaerobically. The reaction time is labeled on the right. For the reaction with dithionite, Dph1–Dph2 was mixed with eEF2 and carboxy-14C-SAM, and dithionite was added to initiate the reaction. For the reaction with Dph3/Cbr1/NADH, Dph1-Dph2 was mixed with Dph3, Cbr1, eEF2, and carboxy-14C-SAM. NADH was added to initiate the reaction. (top) Autoradiography showing labeled eEF2 product. (bottom) eEF2 stained with Coomassie blue (CBB). (C) X-band continuous wave (CW) EPR signal of aerobically purified Dph1–Dph2 with or without dithionite obtained at 12 K. (D) X-band CW EPR signal of anaerobically purified Dph1–Dph2 with or without dithionite obtained at 12 K.

Yugang Zhang, Dan Su, Boris Dzikovski, Sean H. Majer, Rachael Coleman, Siddarth Chandrasekaran, Michael K. Fenwick, Brian R. Crane, Kyle M. Lancaster, Jack H. Freed (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY)
Hening Lin (Department of Chemistry and Chemical Biology and Howard Hughes Medical Institute, Cornell University, Ithaca, NY)

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

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