Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

PDB Reference: bacterioferritin, 2htn, r2htnsf.Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 Å resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on 'as-isolated' E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.

Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine-synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T 0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-b -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.

Generating new carbon-carbon (C-C) bonds in an enantioselective way is one of the big challenges in organic synthesis. Aldolases are a natural tool for stereoselective C-C bond formation in a green and sustainable way. This review will focus on thermophilic aldolases in general and on dihydroxyacetone phosphate-dependent aldolases in particular. Biochemical properties and applications for synthesis of rare sugars and carbohydrates will be discussed.

Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 59 end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His 6 -tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA.