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The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states.

The NDP-forming acyl-CoA synthetases (ACDs) catalyze the conversion of various CoA thioesters to the corresponding acids, conserving their chemical energy in form of ATP. The ACDs are the major energy-conserving enzymes in sugar and peptide fermentation of hyperthermophilic archaea. They are considered to be primordial enzymes of ATP synthesis in the early evolution of life. We present the first crystal structures, to our knowledge, of an ACD from the hyperthermophilic archaeon Candidatus Korachaeum cryptofilum. These structures reveal a unique arrangement of the ACD subunits alpha and beta within an α 2 β 2 -heterotetrameric complex. This arrangement significantly differs from other members of the superfamily. To transmit an activated phosphoryl moiety from the Ac-CoA binding site (within the alpha subunit) to the NDP-binding site (within the beta subunit), a distance of 51 Å has to be bridged. This transmission requires a larger rearrangement within the protein complex involving a 21-aa-long phosphohistidinecontaining segment of the alpha subunit. Spatial restraints of the interaction of this segment with the beta subunit explain the necessity for a second highly conserved His residue within the beta subunit. The data support the proposed four-step reaction mechanism of ACDs, coupling acyl-CoA thioesters with ATP synthesis. Furthermore, the determined crystal structure of the complex with bound Ac-CoA allows first insight, to our knowledge, into the determinants for acyl-CoA substrate specificity. The composition and size of loops protruding into the binding pocket of acyl-CoA are determined by the individual arrangement of the characteristic subdomains.X-ray structure | metabolic energy conversion | protein dynamics | acyl-coenzyme A thioester | superfamily N DP-forming acyl-CoA synthetases (ACDs) catalyze the conversion of acyl-CoA thioesters to the corresponding acids and couple this reaction with the synthesis of ATP via the mechanism of substrate-level phosphorylation. ACDs have been studied in detail in hyperthermophilic archaea, where they function as the major energy-conserving enzymes in the course of anaerobic sugar and peptide fermentation (1-4). It is believed that ACDs represent a primordial mechanism of ATP synthesis in the early evolution of life. ACDs were found in all acetate (acid)-forming archaea (5, 6) and in the eukaryotic parasitic protists Entamoeba histolytica (7) and Giardia lamblia (8), but they have not been found in acetate-forming bacteria. In bacteria, with the exception of Chloroflexus (9), the conversion of inorganic phosphate and the thioester acetyl (Ac)-CoA to acetate and ATP is catalyzed by two enzymes, phosphate Ac-transferase and acetate kinase (10).

Reduced inorganic sulfur compounds like hydrogen sulfide, sulfur, or thiosulfate are attractive prokaryotic energy sources, and their oxidation to sulfuric acid is one of the major reactions of the global sulfur cycle as shown for thiosulfate (Equation 1).Oxidation of inorganic sulfur compounds to sulfate is mainly mediated by various specialized aerobic chemotrophic and anaerobic phototrophic prokaryotes, bacteria and archaea (1-3). Two different modes for bacteria have been proposed recently; one as present in e.g. the anaerobic phototrophic sulfur oxidizing bacterium Allochromatium vinosum involves the reverse acting dissimilatory sulfite dehydrogenase (DsrAB) (Equation 2) which is with 13 other proteins encoded by the dsr operon (4). The product sulfite is subsequently oxidized to sulfate by adenosine 5Ј-phosphosulfate reductase or sulfite:acceptor oxidoreductase (5).The other mode as present in e.g. the aerobic facultative chemotrophic bacterium Paracoccus pantotrophus (6) involves sulfane dehydrogenase SoxCD, which is together with 14 other proteins encoded by the sox operon in this strain. SoxCD is an ␣ 2 ␤ 2 heterotetrameric complex of the molybdoprotein SoxC and the hybrid di-heme cytochrome c like protein SoxD. This sulfane dehydrogenase (formerly designated sulfur dehydrogenase (7)) is a key enzyme of the sulfuroxidizing (Sox) 3 enzyme system and catalyzes the oxidation of protein-bound sulfane-sulfur (oxidation state Ϫ1) to sulfone (oxidation state ϩ5) in a six-electron transfer reaction (8, 9) (Equation 3). SoxZY-SThe current model of the Sox reaction cycle involves sequential activity of four different periplasmic proteins SoxXA, SoxB, SoxYZ, and SoxCD ( Fig. 1) (2, 9). The four proteins oxi-* This work was supported by Deutsche Forschungsgemeinschaft Grants Fr318/10-1 and Sche545/9-1. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Fig. 1

Five experiments have been designed to be used for teaching macromolecular crystallography. The three proteins used in this tutorial are all commercially available; they can be easily and reproducibly crystallized and mounted for diffraction data collection. For each of the five experiments the raw images and the processed data of a sample diffraction data set as well as the refined coordinates and phases are provided for teaching the steps of data processing and structure determination.