1). source of is glucose and the major metabolic end products are lactate, acetate, hydrogen and glycerol (Chapman et al., 1985; Paget and Lloyd, 1990; Kulda, 1999). A key intermediate of metabolism is usually pyruvate which is usually either reduced to lactate in the cytosol by lactate dehydrogenase (Kulda, 1999) or imported into the hydrogenosome, a mitochondrion-related organelle (Lindmark and Mller, 1973; Carlton et al., 2007). There, it is decarboxylated to acetyl-CoA by pyruvate:ferredoxin oxidoreductase (PFOR) which uses ferredoxin as oxidant cofactor. Reduced ferredoxin, in turn, transfers electrons to hydrogenase resulting in the formation of hydrogen. In contrast to (Clark et al., 2007), does not have a bifunctional alcohol/aldehyde dehydrogenase, such as alcohol dehydrogenase 2 in succinyl-CoA synthetase and acetate:succinate CoA-transferase (ASCT) (Lindmark and Mller, 1973; Hesperidin van Grinsven et al., 2008), thereby releasing acetate which is not further utilised. Nevertheless, ethanol is usually a metabolic end product of metabolism (Ellis et al., 1992), albeit Hesperidin of comparably minor importance. Arguably, the most probable source of ethanol in is usually acetaldehyde which is usually reduced by NADP-dependent alcohol dehydrogenase, also termed alcohol dehydrogenase 1 (ADH-1) (Leitsch et al., 2012). It was hypothesised that acetaldehyde is usually formed by PFOR as a side product under anaerobic condition (Leitsch et al., 2012) as described for PFOR Rtn4rl1 from (Ma et al., 1997). In accordance with this hypothesis, ethanol is only formed under anaerobic condition (Ellis et al., 1992). Further, expression of this enzyme is usually down-regulated in some metronidazole-resistant clinical isolates (Leitsch et al., 2012) which display higher levels of intracellular oxygen (Yarlett et al., 1986) and strongly decreased ethanol production rates (Ellis et al., 1992). The conspicuously high expression of ADH-1 in and used for biochemical characterisation, including determination of substrate specificity and kinetic parameters, and identification of inhibitors. 2.?Materials and methods 2.1. Strains and cell culture The strains used were described previously (Leitsch et al., 2012). KV1 (ATCC 30924) was obtained from Caroline Frey (University of Berne, Switzerland) and WB clone C6 (ATCC 50803) was obtained from Norbert Mller (University of Berne, Switzerland). HM-1:IMSS (ATCC30459) had been in our possession Hesperidin before start of this study. was grown as described (Leitsch et al., 2012) in trypticase, yeast extract, maltose (TYM) medium (Diamond, 1957). and were grown in TYI-S-33 medium (Diamond et al., 1978), and was grown in Keisters modified TYI-S-33 medium (Keister, 1983). Cultures were routinely grown in 40?ml culture polystyrene flasks (BD Biosciences). Trypticase was purchased from BD Biosciences and yeast extract was purchased from Merck Chemicals. 2.2. Recombinant expression of ADH-1 The gene (“type”:”entrez-nucleotide”,”attrs”:”text”:”XM_001580551″,”term_id”:”154415150″,”term_text”:”XM_001580551″XM_001580551) was amplified from genomic DNA of strain G3 using the primers 5-TAC GTA CGC ATA TGA CAT TCG AAC TTC CAA AG-3 (forward) and 5-TCA TCC AGG GAT CCT TAG TGA TGG TGA TGG TGA TGA AGC TTG TCG TTG TAT TCG ATG-3 (reverse). PCR fragments were ligated into pET-17b (Novagen) NdeI (forward primer) and BamHI (reverse primer) restriction sites. Expression of recombinant ADH-1 was performed in BL21-AI (Invitrogen) according to the manufacturers protocol. The reverse primer encodes a hexahistidine tag for isolation on NiCNTA spin columns (Qiagen). 2.3. Measurements of ADH-1 activity with the purified recombinant enzyme If not indicated otherwise, reduction of acetone and acetaldehyde was measured at 37?C in 100?mM potassium phosphate buffer pH 6.25, 200?M NADPH, and 1?g?ml?1 recombinant ADH-1 by determining oxidation of NADPH at formation of pentaaqua(thiocyanato-for 5?min. Pellets were washed once in 20?ml 1??PBS followed by another round of centrifugation at 800for 5?min. Cell pellets were resuspended in 500?l of 100?mM potassium phosphate buffer pH 6.25 and lysed with 25 strokes in a Dounce homogeniser. Cell debris and large organelles were removed by centrifugation at 20,000for 10?min. Protein concentrations of lysates were measured using Bradford assay. Enzyme activity was measured in 100?mM potassium phosphate pH 6.25 after adding cell extract (10?g?protein/ml assay buffer) and 200?M NADPH, as oxidation of NADPH at gene (“type”:”entrez-nucleotide”,”attrs”:”text”:”XM_001580551″,”term_id”:”154415150″,”term_text”:”XM_001580551″XM_001580551) was amplified by PCR from genomic DNA and cloned into a pET17-b expression vector. Recombinant ADH-1 was expressed and used for the determination of the enzymatic Hesperidin parameters with the substrates acetaldehyde, acetone, and 2-propanol (Table 1)..
