Transbilayer flipping of glycerophospholipids in the endoplasmic reticulum (ER) is a key feature of membrane biogenesis. Flipping appears to be an ATP-independent, bidirectional process facilitated by specific proteins or flippases. Although a phospholipid flippase has yet to be identified, evidence supporting the existence of dedicated flippases was recently obtained through biochemical reconstitution studies showing that certain chromatographically resolved fractions of detergent-solubilized ER proteins were enriched in flippase activity, whereas others were inactive. We now extend these studies by describing two convenient assays of flippase activity utilizing fluorescent phospholipid analogues as transport reporters. We use these assays to show that (i) proteoliposomes generated from a flippase-enriched Triton X-100 extract of ER can flip analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine; (ii) flipping of all three phospholipids is likely due to the same flippase(s) rather than distinct, phospholipid-specific transport proteins; (iii) functional flippases represent approximately 1% (w/w) of ER membrane proteins in the Triton extract; and (iv) glycerophospholipid flippase activity in the ER can be attributed to two functionally distinct proteins (or classes of proteins) defined by their sensitivity to the cysteine and histidine modification reagents N-ethylmaleimide and diethylpyrocarbonate, respectively. Analyses of the N-ethylmaleimide-sensitive class of flippase activity revealed that the functionally critical sulfhydryl group in the flippase protein is buried in a hydrophobic environment in the membrane but becomes reactive on extraction of the protein into Triton X-100. This observation holds considerable promise for future attempts to isolate the flippase via an affinity approach.
Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonas putida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.
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