Cyclic AMP is a major trigger of the differentiation process of Trypanosoma brucei, a bloodstream parasite causing sleeping sickness. Its generation in trypanosomes is accomplished by a unique battery of membrane-bound adenylate cyclases (ACs). We have determined the high-resolution X-ray structures of the catalytic domains of two trypanosomal ACs (tACs), GRESAG4.1 and GRESAG4.3. The tAC domains are structurally highly related to the AC domains of higher eukaryotes, but also comprise a highly conserved structural element near the active site, the D-subdomain. A cavity below the D-subdomain might correspond to an allosteric regulator site as indicated by the stereospeci®c binding of a single (2S,3S)-1,4-dimercapto-2,3-butanediol molecule. In three different crystal forms, the tAC domains are exclusively observed in a monomeric, catalytically inactive state. Biochemical analysis and the mutagenesis pro®le of GRESAG4.1 con®rmed a common catalytic mechanism of tACs that involves transient dimerization of the AC domain. A low dimerization tendency might play a regulatory role in T.brucei if the activation of tACs is similarly driven by ligand-induced dimerization as in membrane-bound guanylate cyclases.

A novel, 68 amino acid long flavoprotein called dodecin has been discovered in the proteome of Halobacterium salinarum by inverse structural genomics. The 1.7 A crystal structure of this protein shows a dodecameric, hollow sphere-like arrangement of the protein subunits. Unlike other known flavoproteins, which bind only monomeric flavin cofactors, the structure of the dodecin oligomer comprises six riboflavin dimers. The dimerization of these riboflavins along the re-faces is mediated by aromatic, antiparallel pi staggering of their isoalloxazine moieties. A unique aromatic tetrade is formed by further sandwiching of the riboflavin dimers between the indole groups of two symmetry-related Trp36s. So far, the dodecins represent the smallest known flavoproteins. Based on the structure and the wide spread occurrences in pathogenic and soil eubacteria, a function in flavin storage or protection against radical or oxygenic stress is suggested for the dodecins.

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Adenylate cyclases (ACs) are involved in signal transduction by generating the second messenger, cAMP. In Trypanosoma brucei, 3 H ,5 H -cyclic adenosine monophosphate (cAMP) controls the life cycle of this unicellular parasite. cAMP is generated by a class of adenylate cyclases which are either constitutively (GRESAG4.1±4.3) or transiently expressed (ESAG4) during the life cycle. Unlike mammalian ACs, the trypanosomal ACs have a simple topology comprising of a large extracellular region, a transmembrane helix and a cytosolic catalytic region. Two orthorhombic crystal forms of the catalytic AC domain of GRESAG4.1 (residues Ala884±Thr1132) were generated by the hanging-drop vapour-diffusion method. X-ray diffraction data from GRESAG4.1 crystals were collected at 1.9 A Ê resolution using synchrotron radiation. Furthermore, two heavymetal derivative data sets were collected from crystal form A; heavyatom sites were subsequently located in difference Patterson maps.

Alkylhydroperoxide reductases (AhpR, E.C. 1.6.4.x) are essential for the oxygen tolerance of aerobic organisms, converting otherwise toxic hydroperoxides of lipids or nucleic acids to their corresponding alcohols. The AhpF component (521 amino-acid residues, 56.2 kDa) belongs to the family of pyridine nucleotide±disul®de oxidoreductases and channels electrons from NAD(P)H via a series of disul®des towards the AhpC component, which ®nally reduces the hydroperoxide substrates. Crystals of the proteolytically truncated AhpF component (residues Asn208±Ala521) of the alkyl hydroperoxide reductase from Escherichia coli were grown under oxidizing conditions. The crystals belong to space group P3 2 21, with unit-cell parameters a = 60.4, c = 171.8 A Ê . X-ray diffraction data were collected to 1.9 A Ê resolution using synchrotron radiation. A molecularreplacement solution was found using the structure of thioredoxin reductase from Arabidopsis thaliana as a search model.