No description available.

Short/branched chain acyl-CoA dehydrogenase (SBCAD), a member of the acyl-CoA dehydrogenase (ACD) family of enzymes, catalyzes the oxidation of branched chain fatty acids and the branched chain amino acids isoleucine and valine. This research project focuses on expression studies of the SBCAD gene. Northern blot analysis detected two SBCAD mRNA species of 2.7 and 6.5 kb in various human tissues and cell types. A single 4.1 and 2.0 kb SBCAD message was detected in rat and pig tissues, respectively, revealing a species difference in SBCAD mRNA size. Studies of human and rat SBCAD tissue-specificity and relative abundance, at both the RNA and protein levels, identified liver and kidney as the tissues with the highest levels of SBCAD expression, establishing a unique tissue-specific expression pattern that is not seen among the other members of the ACD family. Furthermore, a fetal and adult difference in SBCAD expression was observed in human kidney, suggesting that the SBCAD gene may be developmentally regulated in some tissues. Finally, an attempt was made to isolate and characterize the SBCAD promoter region in order to provide valuable data for future SBCAD promoter studies.

Biocatalysts are increasingly prevalent in the large-scale synthesis of enantiomerically pure compounds. However, many sought-after reactions lack a suitable enzymatic production route. This work describes the development of a novel amine dehydrogenase through the application of directed evolution altering the substrate specificity of an existing leucine dehydrogenase scaffold. Eleven rounds of directed evolution completely altered the enzyme’s specificity and successfully created amination activity. The resulting amine dehydrogenase asymmetrically catalyzes methyl isobutyl ketone and free ammonia to 1, 3-dimethyl butyl amine. The enantioselectivity of the wild-type enzyme was maintained despite the drastic changes to the binding pocket and yielded (R)-1,3-DMBA with nearly complete conversion making it an attractive catalyst in the synthesis of chiral amines. This was the first example of a cofactor-dependent amine dehydrogenase capable of selectively synthesizing chiral amines from a prochiral ketone and free ammonia. Additionally, knowledge gained altering the specificity of the leucine dehydrogenase scaffold was applied to an analogous phenylalanine dehydrogenase scaffold allowing for rapid evolution of novel activity. A single mutational library resulted in a second amine dehydrogenase with enhanced activity toward significantly different substrates, while maintaining comparable conversion and enantioselectivity. These two scaffolds provide examples of the broad applicability of the identified mutations in creating amine dehydrogenase activity.

This thesis presents evidence for a novel mechanism of regulation of pyruvate dehydrogenase (PDH) kinase by fatty acids and also results of a study of muscle triacylglycerol concentration. In animals regulation of PDH complex activity is central to the selection of respiratory fuels and to the conservation of glucose during carbohydrate deprivation. The principal means of regulation of PDH complex is interconversion of phosphorylated (inactive) and dephosphorylated (active) forms effected by PDH kinase and PDH phosphatase. Earlier in vitro studies by others had identified both shorter term (min) and longer term (hours) mechanisms of activation of PDH kinase by fatty acid. In the present study PDH kinase activity (as measured by rates of ATP-dependent inactivation of PDH complex in extracts) was shown to be increased when rat heart mitochondria were incubated with palmitoyl-L-carnitine [PC] (and other CoA utilising respiratory substrates). The activation of PDH kinase persisted through removal of respiratory substrate following incubation with CCCP. A comparable effect of PC was also demonstrable in heart mitochondria from 48h-starved rats (i.e. the mechanism may be distinct from that which increases PDH kinase activity in starvation). Rates of ATP-dependent inactivation of PDH complex were also increased when extracts of rat heart mitochondria were incubated with palmitoyl-CoA (PCoA); the increase was comparable with that seen on incubation of intact mitochondria with PC. The PC effect in intact mitochondria and the PCoA effect in mitochondrial extracts may not be identical as PCoA further increased PDH kinase activity in extracts from mitochondria incubated with PC. Rates of incorporation of ³²P from [γ-<sup>32</supP]ATP into PDH complex were unaltered by pnor incubation of mitochondria with PC or by pnor incubation of mitochondrial extracts with PCoA. Three lines of evidence confirmed that the effect of PC to accelerate ATP-dependent inactivation involved phosphorylation of the PDH complex (viz; use of a non-phosphorylatmg ATP analogue; use of known inhibitors of PDH kinase; and use of known activators/inhibitors of PDH phosphatase). Earlier studies had shown that phosphorylation in punfied bovine and porcine PDH complexes is half site (involves only one α-chain in E1 (α2β2) and had suggested that phosphorylation in rat heart complex may be full site (i.e. involves both α-chains). The present study suggests the possibility that elevation of fatty acyl CoA under slaughter house conditions might be a determinant of half site phosphorylation. A method was developed and evaluated for measurement of triacylglycerol in rat soleus muscle strips with the object of investigating factors that may regulate triacylglycerol synthesis in this muscle. This study was abandoned because, although the method was highly reproducible, great variation was found in the triacylglycerol concentration of individual muscles suggesting the possibility of variable contamination with small amounts of adipose tissue.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed on Aug. 20, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2008.

Thesis (M.S.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains viii, 92 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (M.S.)--Brock University, 2009. / Includes bibliographical references (leaves 69-85).

Thesis (M.D. with Distinction in Research) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Partial embargo. Vita. Bibliography: 44-46