The reactions were analysed with an ABI 310 (Applied Biosystems) or on an ABI 377 (Applied Biosystems) in which case Longranger Single Packs (Cambrex Bio Science, Rockland, Inc., Rockland, ME) were used. Sequence analysis Nucleotide sequences were analysed with computer programs based on those of Devereux et al. [16]. Sequence alignments were performed by using the Blast
programs [17] at the server of the National Center for Biotechnology Information, Bethesda, Md., USA http://www.ncbi.nlm.nih.gov/blast/. Multiple sequence alignments and Epoxomicin purchase construction of the bootstrap tree were performed using ClustalX2.0 [18] Production of recombinant LadA Derivatives of the expression vector pQE32 containing wild type and mutated versions of ladA were transformed to E. coli M13 cells
(Qiagen). Transformation and purification of the recombinant proteins Caspase Inhibitor VI cost using Ni-agarose (Qiagen) was performed according to the supplier’s instructions. Enzyme assays All enzyme assays were performed at 20°C. Dehydrogenase activities were determined using 100 mM glycine pH 9.6, 0.4 mM NAD+ and 100 mM substrate. Reductase activities were determined using 50 mM sodium phosphate pH 7.6, 0.2 mM NADH and 100 mM substrate. Absorbance changes at 340 nm (ε = 6.22 mM-1 cm-1) were measured on a Unicam UV-1 spectrophotometer (Spectronic Unicam, Rochester, NY). Sheep liver SDH was obtained from selleck products Sigma (S3764). Modelling Models of A. niger LadA and XdhA structures were generated using the SWISS-MODEL program http://swissmodel.expasy.org//SWISS-MODEL.html[19–21] with a crystal structure of D-sorbitol dehydrogenase (Protein Data Bank code: 1PL6). In this structure human D-sorbitol dehydrogenase is in complex with the cofactor NAD and an inhibitor [12]. The models were represented using the software package PYMOL [22]. Site-directed mutagenesis Site directed
mutagenesis was performed using the Quik Change protocol (Stratagene, La Jolla, Calif.). Two complementary oligonucleotides of 30–34 nucleotides were designed for each mutation, carrying the mutation in the middle of the oligonucleotide. PCR mixtures contained 50 ng of DNA template, 125 ng of each oligonucleotide, 1 μl of a 10 mM dNTP stock, 5 μl of 10× pfu buffer, and sterile water to a total volume of 24 μl. Before the start of the PCR, 1 μl of Epothilone B (EPO906, Patupilone) pfu DNA polymerase (Stratagene) was added. The reaction parameters were: denaturation of the DNA for 5 min at 95°C, followed by 16 cycles of 30 s denaturation (95°C), 1 min annealing (56°C) and 15 min amplification (68°C). The product was incubated for 4 h with DpnI at 37°C. This enzyme degrades methylated (template) DNA but not the DNA amplified during the PCR. Acknowledgements We would like to thank M. Pail and A. Wiebenga for technical assistance and J.M. van Aken for sequence analysis. LR was supported by the council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW).