Indiana University-Purdue University Indianapolis (IUPUI) / Seven human alcohol dehydrogenase genes (which encode the primary enzymes involved in alcohol metabolism) are grouped into classes based on function and sequence identity. While the Class I ADH isoenzymes contribute significantly to ethanol metabolism in the liver, Class IV ADH isoenzymes are involved in the first-pass metabolism of ethanol. It has been suggested that the ability to efficiently oxidize ethanol occurred late in primate evolution. Kinetic data obtained from the Class I ADH isoenzymes of marmoset and brown lemur, in addition to data from resurrected ancestral human Class IV ADH isoenzymes, supports this proposal--suggesting that two major events which occurred during primate evolution resulted in major adaptations toward ethanol metabolism. First, while human Class IV ADH first appeared 520 million years ago, a major adaptation to ethanol occurred very recently (approximately 15 million years ago); which was caused by a single amino acid change (A294V). This change increases the catalytic efficiency of the human Class IV enzymes toward ethanol by over 79-fold. Secondly, the Class I ADH form developed 80 million years ago--when angiosperms first began to produce fleshy fruits whose sugars are fermented to ethanol by yeasts. This was followed by the duplication and divergence of distinct Class I ADH isoforms--which occurred during mammalian radiation. This duplication event was followed by a second duplication/divergence event which occurred around or just before the emergence of prosimians (some 40 million years ago). We examined the multiple Class I isoforms from species with distinct dietary preferences (lemur and marmoset) in an effort to correlate diets rich in fermentable fruits with increased catalytic capacity toward ethanol oxidation. Our kinetic data support this hypothesis in that the species with a high content of fermentable fruit in its diet possess greater catalytic capacity toward ethanol.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) is the ninth member of the Ca+2-dependent mammalian proprotein convertase super family of serine endoproteases that is structurally related to the bacterial subtilisin and yeast kexin enzymes. It plays a critical role in the regulation of lipid metabolism and cholesterol homeostasis by binding to and degrading low-density lipoprotein-receptor (LDL-R) which is responsible for the clearance of circulatory LDL-cholesterol from the blood. Owing to this functional property, there is plenty of research interest in the development of functional inhibitors of PCSK9 which may find important biochemical applications as therapeutic agents for lowering plasma LDL-cholesterol. The catalytic domain of PCSK9 binds to the EGF-A domain of LDL-R on the cell surface to form a stable complex and re-routes the receptor from its normal endosomal recycling pathway to the lysosomal compartments leading to its degradation. Owing to these findings, we propose that selected peptides from PCSK9 catalytic domain, particularly its disulphide (S-S) bridged loop1 323-358 and loop2 365-385, are likely to exhibit strong affinity towards the EGF-A domain of LDL-R. Several regular peptides along with corresponding all- dextro and retro-inverse peptides as well as the gain-of-function mutant variants were designed and tested for their regulatory effects towards LDL-R expression and PCSK9-binding in human hepatic HepG2 and mouse hepatic Hepa1c1c7 cells. Our data indicated that disulfide bridged loop1-hPCSK9323-358 and its H357 mutant as well as two short loop2-hPCSK9372-380 and its Y374 mutant peptides modestly promote the LDL-R protein levels. Our study concludes that specific peptides from the PCSK9 catalytic domain can regulate LDL-R and may be useful for development of novel class of therapeutic agents for cholesterol regulation.

The proven ability to ferment Saccharomyces cerevisiae on a large scale presents an attractive target for producing chemicals and fuels from sustainable sources. Efficient and predominant carbon flux through to ethanol is a significant engineering issue in the development of this yeast as a multi-product cell chassis used in biorefineries. In order to evaluate diversion of carbon flux away from ethanol, combinatorial deletions were investigated in genes encoding the six isozymes of alcohol dehydrogenase (ADH), which catalyse the terminal step in ethanol production. The scarless, dominant and counter- selectable amdSYM gene deletion method was optimised for generation of a combinatorial ADH knockout library in an industrially relevant strain of S. cerevisiae. Current understanding of the individual ADH genes fails to fully evaluate genotype-by-genotype and genotype-by-environment interactions: rather, further research of such a complex biological process requires a multivariate mathematical modelling approach. Application of such an approach using the Design of Experiments (DoE) methodology is appraised here as essential for detailed empirical evaluation of complex systems. DoE provided empirical evidence that in S. cerevisiae: i) the ADH2 gene is not associated with producing ethanol under anaerobic culture conditions in combination with 25 g l-1 glucose substrate concentrations; ii) ADH4 is associated with increased ethanol production when the cell is confronted with a zinc-limited [1 μM] environment; and iii) ADH5 is linked with the production of ethanol, predominantly at pH 4.5. A successful metabolic engineering strategy is detailed which increases the product portfolio of S. cerevisiae, currently used for large-scale production of bioethanol. Heterologous expression of the cytochrome P450 fatty acid peroxygenase from Jeotgalicoccus sp., OleTJE, fused to the RhFRED reductase from Rhodococcus sp. NCIMB 978 converted free fatty acid precursors to C13, C15 and C17 alkenes (3.81 ng μl-1 total alkene concentration).