Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis

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Abstract

<p class="first" id="P1">Diphthamide biosynthesis involves a carbon-carbon bond forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a C-S bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an Fe-C bond between ACP and the enzyme’s [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme. </p><p id="P2">An unusual radical enzyme forms an iron-carbon bond as the first step in modification of a protein side chain. </p>

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Optimal description of a protein structure in terms of multiple groups undergoing TLS motion.

Jay Painter, Ethan A. Merritt (2006)

A single protein crystal structure contains information about dynamic properties of the protein as well as providing a static view of one three-dimensional conformation. This additional information is to be found in the distribution of observed electron density about the mean position of each atom. It is general practice to account for this by refining a separate atomic displacement parameter (ADP) for each atomic center. However, these same displacements are often described well by simpler models based on TLS (translation/libration/screw) rigid-body motion of large groups of atoms, for example interdomain hinge motion. A procedure, TLSMD, has been developed that analyzes the distribution of ADPs in a previously refined protein crystal structure in order to generate optimal multi-group TLS descriptions of the constituent protein chains. TLSMD is applicable to crystal structures at any resolution. The models generated by TLSMD analysis can significantly improve the standard crystallographic residuals R and R(free) and can reveal intrinsic dynamic properties of the protein.

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Diphthamide biosynthesis requires an Fe-S enzyme-generated organic radical

Yang Zhang, Xuling Zhu, Andrew T. Torelli … (2010)

Summary Archaeal and eukaryotic translation elongation factor 2 contain a unique posttranslationally modified histidine residue called “diphthamide”, the target of diphtheria toxin. The biosynthesis of diphthamide were proposed to involve three steps, with the first step being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosylmethionine (SAM). However, details of the biosynthesis have remained unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulfur cluster enzyme, Dph2. Dph2 is a homodimer and each monomer contains a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5′-deoxyadenosyl radical. Instead, it breaks the Cγ,Met-S bond of SAM and generates a 3-amino-3-carboxylpropyl radical. This work suggests that Pyrococcus horikoshii Dph2 represents a novel SAM-dependent [4Fe-4S]-containing enzyme that catalyzes unprecedented chemistry.

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Nitrosomonas europaeacytochrome P460 is a direct link between nitrification and nitrous oxide emission

Avery Vilbert, Jonathan Caranto, Kyle M Lancaster (2016)

Nitrous oxide (N 2 O) is a potent ozone-depleting greenhouse gas. This work identifies a means by which N 2 O is generated during nitrification, or biological ammonia oxidation. Fertilizer use in agriculture stimulates nitrification, thus increasing the volume of N 2 O emissions worldwide. The results presented herein will inform models and strategies toward optimized, sustainable agriculture. Moreover, these results highlight a rare example of biological N–N bond formation. Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N 2 O). It has been proposed that N 2 O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH 2 OH) quantitatively to N 2 O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH 2 OH to nitrite ( N O 2 − ) under aerobic conditions. Although we observe N O 2 − formation under aerobic conditions, its concentration is not stoichiometric with the NH 2 OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH 2 OH to N 2 O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe III –NH 2 OH adduct of cyt P460 is oxidized to an {FeNO} 6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N–N bond of N 2 O during a bimolecular, rate-determining step. We propose that N O 2 − results when nitric oxide (NO) dissociates from the {FeNO} 6 intermediate and reacts with dioxygen. Thus, N O 2 − is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH 2 OH contributes to NO and N 2 O emissions from nitrifying microorganisms.