Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h –1 L –1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

Background: Berberine bridge enzyme-like proteins are a multigene family in plants.Results: Members of the berberine bridge enzyme-like family were identified as monolignol oxidoreductases.Conclusion: Berberine bridge enzyme-like enzymes play a role in monolignol metabolism and lignin formation.Significance: Our results indicate a novel and unexpected role of berberine bridge enzyme-like enzymes in plant biochemistry and physiology.

Difficulties involved in obtaining a firm diagnosis have led to a variety of terms being used to describe this congenital disease. Diagnosis of agenesis of the dorsal pancreas is inconclusive without demonstration of the absence of the dorsal pancreatic duct. Here we describe the embryological development of the pancreas, the so-far known cases of agenesis of the dorsal pancreas with associated medical problems, and the diagnostic measures to find the right conclusions.

Objective-To study the eVect of a standardised training programme focusing on maintenance of fat free mass during weight reduction by energy reduction in obese children. Design-Randomised trial of physical training programme and dietary advice (group A) versus dietary advice alone (group B).Subjects-Thirty obese children and adolescents (14 group A, 16 group B) participated in the 12 week long programme; 20 children (10 group A, 10 group B) were also reassessed after one year. Measurements-Fat free mass was estimated from the resistance index, obtained by bioelectrical impedance analysis at baseline, after four, eight, and 12 weeks in all subjects, and after one year in 20 subjects. Results-The mean (SD) change in fat free mass was significantly diVerent between the two groups after 12 weeks (group A, 2.68 (3.74) kg; group B, 0.43 (1.65) kg). The change in body weight after one year was inversely correlated with the change in fat free mass after 12 weeks (r = −0.44), as assessed in the 20 subjects. Conclusions-A standardised training programme as used in this study can prevent reduction in fat free mass during weight loss in obese children. Reduction in fat free mass during weight reduction might be a risk factor for regain of weight. (Arch Dis Child 1999;81:426-428) Keywords: training programme; obesity; body composition Obesity is an ongoing problem in paediatric and adolescent health care. In previous work we identified reduction in fat free mass during rapid weight loss by energy reduction as a major factor for later regain in weight.1 Our present study aimed to: evaluate a standardised training programme for maintenance of fat free mass during weight reduction; and study the eVect of changes in fat free mass during weight reduction on the long term outcome. Resistance training has been shown to be eVective in increasing strength in children. 2There is limited information as to whether muscle growth (hypertrophy) can be induced in obese children. SubjectsThirty children consented to participate in the study and were assigned to two groups at random: group A (six boys, eight girls; mean (SD) age, 11.0 (2.5) years; mean standard deviation score for body mass index (BMI-SDS), 5.58 (2.46)) received standardised dietary advice for weight reduction by a dietitian at baseline and after four, eight, and 12 weeks of the study. In addition, subjects participated in a training programme twice weekly. Group B (seven boys, nine girls; mean age, 12.2 (2.7) years; mean BMI-SDS, 5.33 (1.79)) had the dietary intervention only. Methods BODY COMPOSITIONBody composition was estimated from bioelectrical impedance analysis. Measurements were performed at baseline and after four, eight, and 12 weeks. Total body resistance was measured by a bioelectrical impedance analyser (Akern-RJL BIA 101/S) in supine position as described previously.1 Fat free mass was estimated from the resistance index (RI), height, and age of the subject using the equations given by Shaefer et al (fat free mass (kg) = 0.15 + 0.65 × RI + 0.68 × age...