No abstract

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.

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo-and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn 116 , Ser 144 , Tyr 159 , and Lys 163 . GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser 144 , Ser 146 , and Asn 151 are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.Dehydrogenases represent an important class of enzymes in biotechnological processes increasingly used in the chemical or pharmaceutical industry due to their enantio-and stereoselective oxidative and reductive catalytic properties (1-4). A dehydrogenase with a promising catalytic potential is galactitol dehydrogenase (galactitol:NAD ϩ 5-oxidoreductase (GatDH) 3 ), an enzyme originally isolated from a galactitol-utilizing mutant of the bacterium Rhodobacter sphaeroides (5). GatDH is a homotetrameric protein that requires Mg 2ϩ for maintenance of its quaternary structure and enzymatic activity. It catalyzes the dehydrogenation of a variety of polyvalent aliphatic alcohols and polyols to the corresponding ketones and ketoses, respectively, and in the reverse reaction it reduces prochiral ketones with high stereoselectivity yielding the corresponding S-configured secondary alcohols (5-7). Based on these catalytic capabilities, GatDH was used with cofactor recycling (8, 9) for several preparative conversions. Oxidation at C5 of galactitol gave the rare sugar L-tagatose in almost quantitative yields (6). L-Tagatose is a precursor for the synthesis of the glycosidase inhibitor 1-deoxygalactonojirimycin (10) and is a substrate of pyranose 2-oxidase yielding the interesting synthon 5-keto-psicose (11). Similarly, xylitol is oxidized at C4 to give L-xylulose (5). We also demonstrated the preparation of several (R)-1,2-diols by racemic resolution with GatDH as well as the synthesis of several S-configured aliphatic alcohols by reducing corresponding prochiral ketones (7). Furthermore, the suitability of GatDH for conversions with electrochemical cofactor regeneration was demons...

The high-resolution crystal structure of the flavin-dependent monooxygenase (FMO) from the African locust Zonocerus variegatus is presented and the kinetics of structure-based protein variants are discussed. Z. variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. N-Oxygenation of PAs and the accumulation of PA N-oxides within their haemolymph result in two evolutionary advantages for these insects: (i) they circumvent the defence mechanism of their food plants and (ii) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. Here, the crystal structure of the Z. variegatus PA N-oxygenase (ZvPNO), the most active ZvFMO isoform, is reported at 1.6 Å resolution together with kinetic studies of a second isoform, ZvFMOa. This is the first available crystal structure of an FMO from class B (of six different FMO subclasses, A-F) within the family of flavin-dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. This indicates the need for high conservation of the three-dimensional structure for this type of reaction throughout all kingdoms of life. Nevertheless, this structure provides the closest relative to the human enzyme that is currently available for modelling studies. Of note, the crystal structure of ZvPNO reveals a unique dimeric arrangement as well as small conformational changes within the active site that have not been observed before. A newly observed kink within helix α8 close to the substrate-binding path might indicate a potential mechanism for product release. The data show that even single amino-acid exchanges in the substrate-entry path, rather than the binding site, have a significant impact on the specific enzyme activity of the isoforms.

Two new experiments (single isomorphous replacement including anomalousscattering effects and radiation damage-induced phasing) have been designed to complement the five experiments (sulfur single-wavelength anomalous diffraction, multiple-wavelength anomalous diffraction, molecular replacement, ion binding and ligand binding) of the first edition of the previously described tutorial for learning and teaching macromolecular crystallography [Faust, Panjikar, Mueller, Parthasarathy, Schmidt, Lamzin & Weiss (2008). J. Appl. Cryst. 41, 1161-1172]. Furthermore, the tutorial has been re-organized and in part re-written to reflect the comments and suggestions of users. The most significant overhaul was applied to the data-processing part of the tutorial. Nevertheless, the convenient features that all of the utilized proteins used are commercially available and that they can be easily and reproducibly crystallized and mounted for diffraction data collection have been retained. Also, for all of the seven experiments the raw diffraction images and the processed data are provided for illustrating and teaching the steps of data processing and structure determination.