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In NAD(P)-dependent enzymes the coenzyme gives or takes a hydride ion, but how the nicotinamide ring is activated to form the transition state for hydride transfer is not clear. On the basis of ultra-high resolution X-ray crystal structures of liver alcohol dehydrogenase (LADH) in complex with NADH and a number of substrate analogues we proposed that the activation of NADH is an integral part of the enzyme mechanism of aldehyde reduction [R. Meijers, R.J. Morris, H.W. Adolph, A. Merli, V.S. Lamzin, E.S. Cedergren-Zeppezauer, On the enzymatic activation of NADH, The Journal of Biological Chemistry 276(12) (2001) 9316–9321, %U http://www.ncbi.nlm.nih.gov/pubmed/11134046 ; R. Meijers, H.-W. Adolph, Z. Dauter, K.S. Wilson, V.S. Lamzin, E.S. Cedergren-Zeppezauer, Structural evidence for a ligand coordination switch in liver alcohol dehydrogenase, Biochemistry 46(18) (2007) 5446–5454, %U http://www.ncbi.nlm.nih.gov/pubmed/17429946 ]. We observed a nicotinamide with a severely distorted pyridine ring and a water molecule in close proximity to the ring. Quantum chemical calculations indicated that (de)protonation of the water molecule can be directly coupled to activation of NADH for hydride transfer. A systematic search of the Protein Data Bank (PDB) for atoms that come within van der Waals distance of the pyridine ring of the nicotinamide reveals that a large number of NAD(P)-containing protein complexes are involved in electrostatic interactions with the enzymatic environment. Using the deposited diffraction data to analyze the cofactor and its surroundings, we observe several adducts between protein atoms and the pyridine ring that were not previously reported. This further indicates that the enzymatic activation of NAD(P) induced by electrostatic interactions is an essential part of the hydride transfer mechanism.

Abstract: Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), an enzyrrie in the nucleotide metabolism, from the retrovirus Equine Infectious Anemia Virus (EIAV) has been crystilllized from PEG mixtures at neutral pH using a vapour diffusion method. Crystals of native enzyme belong to the space group R32, while crystals formed in the presence of Sr2+ and dUDP, a substrate analogue, belong to the space group P4132 or P4332. Both forms are suitable for high resolution X-ray structure analyses.

A system of novel analytical tools for the analysis of intra- and extra-cellular events on a level of single cells have been developed, comprising equipment for acoustic levitation and piezo-electric flow-through micro-dispensing for nano-volume sample handling and sample preparation. The system has been used to study lipolysis in individual adipocytes using fluorescence imaging detection and shows great promise for future applications such as cell-based secondary high throughput screening (HTS).

Thirty-one and eleven sequences for the photosystem II reaction centre proteins D1 and D2 respectively, were compared to identify conserved single amino acid residues and regions in the sequences. Both proteins are highly conserved. One important difference is that the lumenal parts of the D1 protein are more conserved than the corresponding parts in the D2 protein. The three-dimensional structures around the electron donors tyrosineZ and tyrosineD on the oxidizing side of photosystem II have been predicted by computer modelling using the photosynthetic reaction centre from purple bacteria as a framework. In the model the tyrosines occupy two cavities close to the lumenal surface of the membrane. They are symmetrically arranged around the primary donor P680 and the distances between the centre of the tyrosines and the closest Mg ion in P680 are around 14 A. Both tyrosineZ and tyrosineD are suggested to form a hydrogen bond with histidine 190 from the loop connecting helices C and D in the D1 and D2 proteins, respectively. The Mn cluster in the oxygen evolving complex has been localized by using known and estimated distances from the tyrosine radicals. It is suggested that a binding region for the Mn cluster is constituted by the lumenal ends of helices A and B and the loop connecting them in the D1 protein. This part of the D1 protein contains a large number of strictly conserved carboxylic acid residues and histidines which could participate in the Mn binding. There is little probability that the Mn cluster binds on the lumenal surface of the D2 protein.

A miniaturized analysis system for the studying of living cells and biochemical reactions in microdrops was developed. Cell studies were performed using single adipocytes in 250‐nL drops. Continuous flow‐through droplet dispensers, developed in‐house, were used for additions to the levitated droplet. Addition of b‐adrenergic agonists stimulates the lipolysis in the adipocytes, leading to free fatty acid release and a consequent pH decrease of the surrounding buffer, a change that can be easily followed using a pH‐dependent fluorophore continuously monitored by fluorescence imaging detection. An analytical method using capillary electrophoresis and nanospray mass spectrometry for measurement of the cAMP level in activated single adipocytes are now being developed for future use in combination with the levitation technique. The levitation approach was also employed for the screening of nucleation conditions for macromolecules. Here, the acoustic levitator offers a simplified way to determine the main features of the phase diagram (i.e., precipitation diagram). Using the droplet dispensers, different types and amounts of precipitation agents are injected into the levitated drop, allowing a systematic search for nucleation conditions that is not possible using standard crystallization methods. Once the precipitation diagram has been obtained, optimization using standard methods is employed to grow the crystals.

Ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco is bifunctional (Miziorko, Lorimer, 1983). In photosynthesis, the carboxylase activity of the enzyme catalyzes the condensation of CO2 to ribulose 1,5-bisphosphate (RuBP) to yield two moles of 3-D-phosphoglycerate (PGA). The oxygenase activity, on the other hand, catalyzes the addition of O2 to RuBP to yield PGA and 2-phosphoglycolate. The last reaction results in net loss of fixed CO2. An understanding of the structural basis for these two activities is therefore important for genetic modifications of Rubisco in plants (Sommerville, Ogren, 1982).