ABSTRACT
The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of â¼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/metabolism , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/metabolism , Phenylglyoxal/analogs & derivatives , Sulfolobus acidocaldarius/enzymology , Amino Acid Sequence , Archaeal Proteins/genetics , Benzoin/metabolism , Enzyme Stability , Fatty Acid Synthases/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , NADH, NADPH Oxidoreductases/genetics , Phenylglyoxal/metabolism , Sequence Homology, Amino Acid , Stereoisomerism , Substrate SpecificityABSTRACT
Superoxide dismutases (SODs) are key enzymes for fighting oxidative stress. Helicobacter pylori produces a single SOD (HpSOD) which contains iron. The structure of this antioxidant protein has been determined at 2.4 A resolution. It is a dimer of two identical subunits with one iron ion per monomer. The protein shares 53% sequence identity with the corresponding enzyme from Escherichia coli. The model is compared with those of other dimeric Fe-containing SODs. HpSOD shows significant differences in relation to other SODs, the most important being an extended C-terminal tail. This structure provides a model for closely related sequences from species such as Campylobacter, where no structures are currently known. The structure of extended carboxyl termini is discussed in light of putative functions it may serve.
Subject(s)
Helicobacter pylori/enzymology , Superoxide Dismutase/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Dimerization , Helicobacter pylori/chemistry , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Superoxide Dismutase/metabolismABSTRACT
Single chains of the collagen model polypeptide with sequence (Pro-Pro-Gly)(10), hereafter referred to as (PPG)(10), aggregate to form rod-shaped triple helices. Crystals of (PPG)(10) were grown in the Advanced Protein Crystallization Facility (APCF) both onboard the International Space Station (ISS) and on Earth. The experiments allow the direct comparison of four different crystallization environments for the first time: solution in microgravity ((g), agarose gel in (g, solution on earth, and gel on earth. Both on board and on ground, the crystal growth was monitored by a CCD video camera. The image analysis provided information on the spatial distribution of the crystals, their movement and their growth rate. The analysis of the distribution of crystals reveals that the crystallization process occurs as it does in batch conditions. Slow motions have been observed onboard the ISS. Different to Space-Shuttle experiment, the crystals onboard the ISS moved coherently and followed parallel trajectories. Growth rate and induction time are very similar both in gel and in solution, suggesting that the crystal growth rate is controlled by the kinetics at the interface under the used experimental conditions. These results provide the first data in the crystallogenesis of (PPG)(10), which is a representative member of non-globular, rod-like proteins.
Subject(s)
Collagen/chemistry , Crystallization/methods , Peptides/chemistry , Gels , Sepharose , Solutions , Space Flight , Time Factors , Video Recording , WeightlessnessABSTRACT
Crystals of the collagen-like polypeptide (PPG)(10) were obtained within the Advanced Protein Crystallization Facility on board the International Space Station, during the STS-105/STS-108 mission. The duration of this mission was such to ensure that the crystallization process had reached its end. Crystals were grown both in the presence and in the absence of agarose gel, to compare the quality of the crystals obtained from these different environments. As a result, crystals grown in the absence of agarose on Earth as well as in microgravity showed X-ray diffraction up to 1.15 A. The intensity/sigma ratio was slightly higher for microgravity grown crystals. Crystals grown in agarose gel, both in microgravity and on ground, showed a comparable diffraction power, with a resolution limit of 1.45 A.
