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The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.

The crystal structure of the apo-form of an R-specific alcohol dehydrogenase from Lactobacillus brevis (LB-RADH) was solved and refined to 1.8A resolution. LB-RADH is a member of the short-chain dehydrogenase/reductase (SDR) enyzme superfamily. It is a homotetramer with 251 amino acid residues per subunit and uses NADP(H) as co-enzyme. NADPH and the substrate acetophenone were modelled into the active site. The enantiospecificity of the enzyme can be explained on the basis of the resulting hypothetical ternary complex. In contrast to most other SDR enzymes, the catalytic activity of LB-RADH depends strongly on the binding of Mg(2+). Mg(2+) removal by EDTA inactivates the enzyme completely. In the crystal structure, the Mg(2+)-binding site is well defined. The ion has a perfect octahedral coordination sphere and occupies a special position concerning crystallographic and molecular point symmetry, meaning that each RADH tetramer contains two magnesium ions. The magnesium ion is no direct catalytic cofactor. However, it is structurally coupled to the putative C-terminal hinge of the substrate-binding loop and, via an extended hydrogen bonding network, to some side-chains forming the substrate binding region. Therefore, the presented structure of apo-RADH provides plausible explanations for the metal dependence of the enzyme.

A new NADH oxidase, useful for the regeneration of NAD+, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10-15 U mg(-1). After purification by four chromatographic steps, an activity of 116 U mg(-1) was obtained with 14% yield. Highest activity was from pH 5.5-7 and at 40 degrees C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The Km value for NADH was 24 microM.

The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is a valuable catalyst for the production of chiral alcohols that can be used as synthons in asymmetric syntheses. RADH is a homotetramer with 222 symmetry and a molecular mass of 107 kDa. The recombinant enzyme has been expressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 56.5, b = 85.1, c = 115.4 A, and diffract X-rays to at least 1.8 A resolution. The calculated crystal packing parameter V(M) = 2.59 A(3) Da(-1), corresponding to a solvent content of 52.5% and suggesting that one RADH monomer is contained in the asymmetric unit. The RADH tetramer lies on a special position with its molecular dyads coinciding with the crystallographic twofold axes and with its centre of mass on the origin of the unit cell.

The main stimulant for the use of anabolic agents by competitive athletes is their effect on the quantity, structure, and power of the muscles, although an actual increase in efficiency is most doubtful. This effect has to be purchased with a number of possible side effects including acne or acneiform lesions - an interesting aspect from the dermatologic point of view, since androgens may have an etiopathogenetic significance in this disease. We report on a 23-year-old male patient (competitive javelin thrower) who developed acne fulminans while using anabolics and vitamin B preparations. The temporal correlation between the onset of the skin lesions and the intake of the drugs suggests that the drugs - either alone or in combination - may be responsible for the skin eruptions. We discuss the case in detail referring to the literature.