estrone We compared the mixotrophic growth of mutant DRHB

We compared the mixotrophic growth of mutant DRHB1486 and the wild-type at different light intensities. The mutant grew at a similar rate as the wild-type at a low light intensity but much more slowly at a high light intensity (Fig. 3). Because the slr2094 mutant was unable to grow under photoautotrophic conditions and its mixotrophic growth was inhibited at relatively high light intensities, we hypothesized that certain accumulated intermediates (such as fructose-1,6-biphosphate and sedoheptulose-1,7-bisphosphate) were harmful to cyanobacterial estrone and inhibited the mixotrophic growth of the mutant. We inactivated psbB in the DRHB1486 mutant. The psbB gene encodes the chlorophyll-binding protein CP-47 of photosystem II [21], [22]. The resulted double mutant DRHB1486/psbB::C.K2 was unable to grow under photoautotrophic conditions as the single mutant DRHB1486, but showed significantly increased mixotrophic growth in the light of 25μEm−2s−1 compared with DRHB1486 (Fig. 4). In the presence of DCMU, an inhibitor of photosystem II, the three strains showed similar growth on glucose (Fig. 4). The resumption of mixotrophic growth of the slr2094 mutant by inactivation of psbB supported our hypothesis. Based on this finding, it should be possible to enrich mutant cells defective in photosystem II with an slr2094 null mutation in the future.
Acknowledgements
Introduction Noninsulin-dependent diabetes mellitus (NIDDM), also known as type II diabetes, results from a combination of insulin resistance of target cells (liver, muscle and fat) and relative deficiencies in insulin production from the β-cells of pancreatic islets [1]. The molecular basis of NIDDM is poorly understood, but whatever the pathogenesis behind this serious condition is, it always manifests itself by hyperglycemia which is caused by reduced entry/utilization of glucose into/by various tissues and increased release of glucose into the circulation by hepatic gluconeogenesis [2], [3], [4]. Consequently, reduction of the gluconeogenic rate in NIDDM should provide treatment for NIDDM patients [5]. Glycolysis and gluconeogenesis (GNG) are intracellular catabolic and anabolic pathways of glucose metabolism, respectively. Although most enzymes in liver are shared by the two pathways, fructose-6-phosphate-1-kinase catalyzes the unidirectional phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate in glycolysis, while fructose-1,6-bisphosphatase (d-fructose 1,6-bisphosphate 1-phosphohydrolase, FBPase) catalyzes the reverse reaction in gluconeogenesis, i.e., the hydrolysis of fructose 1,6-bisphosphate (F1, 6P) to fructose 6-phosphate (F6P) and inorganic phosphate [6]. Therefore FBPase is regarded as one of the key enzymes in gluconeogenesis [7]. FBPase is a homotetramer [subunit Mr = 37,000] with at least two quaternary states referred to as R (active state) and T (less active state). The amino acid sequence of the human liver F1,6BP was recently determined and found to be approximately 90% identical to the porcine kidney enzyme [5], [8], [9]. Each tetramer subunit has an allosteric AMP domain (residues 1–20) and a catalytic FBPase domain (residues 201–335), with the AMP-binding site located approximately 28 Å away from the substrate binding site [10], [11], [12]. X-ray structures of AMP, as well as AMP analogs, complexed to the allosteric binding pocket of human liver FBPase have been solved [13]. The number and nature of the divalent cations required in the human liver enzyme have not yet been fully investigated. One and two Mg2+ ions have been found in the human enzyme, and similar observations have also been reported for the porcine enzyme. The allosteric mechanism has also been investigated. The tetramer can be divided into two dimers, an upper dimer (two subunits) and a lower dimmer (two subunits). A 16–17° rotation of the lower dimer relative to the upper dimer has been described for the porcine FBPase upon binding of AMP to the allosteric binding pocket. The unrotated form is called the R form and the rotated form is called the T form. The only structure reported of FBPase complexed with an allosteric inhibitor is AMP (porcine and human FBPase enzyme) [5], [14].