Derivatives of vitamin B12 are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO2 fixation in acetogenic bacteria1–3. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect, and perform catalysis on the reactive B12 cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes4–12, inspiring speculation regarding the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete ~220 kDa complex that contains all enzymes responsible for B12-dependent methyltransfer, namely the corrinoid iron-sulfur protein (CFeSP) and its methyltransferase (MeTr) from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection, and catalytic steps of B12-dependent methyltransfer. In addition, the structures capture B12 at multiple locations between its “resting” and catalytic positions, allowing visualisation of the dramatic protein rearrangements that enable methyltransfer and identification of the trajectory for B12 movement within the large enzyme scaffold. The structures are also presented alongside in crystallo UV-vis spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and L-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes L-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.crystal structure | molecular dynamics | epothilone | natural product

Ni-Fe containing enzymes are involved in the biological utilization of carbon monoxide, carbon dioxide, and hydrogen. Interest in these enzymes has increased in recent years due to hydrogen fuel initiatives and concerns over development of new methods for CO 2 sequestration. One Ni-Fe enzyme called carbon monoxide dehydrogenase (CODH) is a key player in the global carbon cycle and carries out the interconversion of the environmental pollutant CO and the greenhouse gas CO 2 . The Ni-Fe center responsible for this important chemistry, the C-cluster, has been the source of much controversy, but several recent structural studies have helped to direct the field toward a unifying mechanism. Here we summarize the current state of understanding of this fascinating metallocluster.While enzymes that utilize iron-containing active sites catalyze a wide range of well-known chemical transformations, three remarkable enzymes combine iron and nickel into complex metalloclusters that extend Nature's biochemical toolkit and lie at the heart of fundamental biological processes involving microbial hydrogen utilization and carbon fixation.[NiFe]-hydrogenase can catalyze both H 2 oxidation and evolution in anaerobic microbes to consume or produce protons and electrons, the biological equivalent of the hydrogen fuel cell anode [1,2]. Involved in carbon fixation, the enzyme acetyl-CoA synthase (ACS) contains a Ni-Fe-S active site metal center called the A-cluster that combines carbon monoxide (CO) with a methyl group and coenzyme A (CoA) to form acetyl-CoA, generating a source of carbon and energy for a variety of microbes. CO is often provided to ACS by another Ni-Fe enzyme called carbon monoxide dehydrogenase (CODH), a dimeric enzyme which contains a distinctive Ni-Fe-S metal center termed the C-cluster that carries out the reversible reduction of carbon dioxide (CO 2 ) to CO, the biological equivalent of the water-gas shift reaction and a mechanism for CO 2 utilization. Interest in all three enzymes has increased dramatically in recent years due to renewed attention in the development of hydrogen fuel cells and the design of CO 2 sequestration technologies. While the first X-ray crystal structures of [NiFe]-hydrogenase [3], ACS [4], and CODH [5,6] revealed the overall Corresponding author: Drennan, C.L. (cdrennan@mit.edu,). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. architecture of these complex metallocenters, recent work has aimed to develop an understanding of the mechanisms by which these clusters catalyze their respective reactions. As major advances have been made on the C...

Nickel-containing carbon monoxide dehydrogenases (CODHs) reversibly catalyze the oxidation of carbon monoxide to carbon dioxide and are of vital importance in the global carbon cycle. The unusual catalytic CODH C-cluster has been crystallographically characterized as either a NiFe 4 S 4 or a NiFe 4 S 5 metal center, the latter containing a fifth, additional sulfide that bridges Ni and a unique Fe site. To determine whether this bridging sulfide is catalytically relevant and to further explore the mechanism of the C-cluster, we obtained crystal structures of the 310 kDa bifunctional CODH/acetyl-CoA synthase complex from Moorella thermoacetica bound both with a substrate H 2 O/OH -molecule and with a cyanide inhibitor. X-ray diffraction data were collected from native crystals and from identical crystals soaked in a solution containing potassium cyanide. In both structures, the substrate H 2 O/OH -molecule exhibits binding to the unique Fe site of the C-cluster. We also observe cyanide binding in a bent conformation to Ni of the C-cluster, adjacent the substrate H 2 O/OH -molecule. Importantly, the bridging sulfide is not present in either structure. As these forms of the C-cluster represent the coordination environment immediately before the reaction takes place, our findings do not support a fifth, bridging sulfide playing a catalytic role in the enzyme mechanism. The crystal structures presented here, along with recent structures of CODHs from other organisms, have led us toward a unified mechanism for CO oxidation by the C-cluster, the catalytic center of an environmentally important enzyme.Carbon monoxide dehydrogenases (CODHs) are key enzymes in the global carbon cycle and catalyze the reversible conversion of CO to CO 2 . In some anaerobic bacteria, including the phototroph Rhodospirillum rubrum and the thermophile Carboxydothermus hydrogenoformans, monofunctional Ni-containing CODHs allow these organisms to use CO as their sole carbon and energy source (1, 2). CODH activity accounts for the removal of ∼10 8 tons of CO from the environment every year (3). Acetogenic bacteria, including well-characterized Moorella thermoacetica, couple CODH-catalyzed CO 2 reduction with acetyl-CoA synthesis in the bifunctional enzyme complex CODH/ acetyl-CoA synthase (ACS) as part of the Wood-Ljungdahl carbon fixation pathway (4-7). Briefly, in the "eastern" branch of the pathway, one molecule of CO 2 is reduced to a methyl group in a series of folate-dependent steps. The methyl group is then transferred from methyltetrahydrofolate to the corrinoid ironsulfur protein (CFeSP) by methyl-H 4 folate:CFeSP methyltransferase (MeTr). In the "western" branch (Scheme 1), where Nicontaining CODH/ACS is the principal player, a second molecule of CO 2 is reduced to a CO intermediate by the CODH active site C-cluster. CO then travels ∼70 Å through a remarkable tunnel within the enzyme to the ACS active site A-cluster (8-12), where it is combined with the CFeSP-derived methyl group and coenzyme A to form acetyl-CoA. Acetyl-CoA c...

The archaeal enzyme geranylgeranyl reductase (GGR) catalyzes hydrogenation of carbon-carbon double bonds to produce the saturated alkyl chains of the organism's unusual isoprenoid-derived cell membrane. Enzymatic reduction of isoprenoid double bonds is of considerable interest both to natural products researchers and to synthetic biologists interested in the microbial production of isoprenoid drug or biofuel molecules. Here we present crystal structures of GGR from Sulfolobus acidocaldarius, including the structure of GGR bound to geranylgeranyl pyrophosphate (GGPP). The structures are presented alongside activity data that depict the sequential reduction of GGPP to H6GGPP via the intermediates H2GGPP and H4GGPP. We then modified the enzyme to generate sequence variants that display increased rates of H6GGPP production or are able to halt the extent of reduction at H2GGPP and H4GGPP. Crystal structures of these variants not only reveal the structural bases for their altered activities; they also shed light onto the catalytic mechanism employed.