The adult human liver flavin-containing monooxygenases (HLFMO) remains as one of the key classes of important human monooxygenases that have not been studied in a systematic and comprehensive manner. It is now recognized that one form of the enzyme, HLFMO3, is the primary enzyme present in adult human liver and is receiving increased attention. Due to the recognition that mutant HLFMO3 may be responsible for trimethylaminuria (i.e., 'fish-odor syndrome') and other human diseases, it is imperative that this enzyme be studied. The long term goal of our research is to understand the physiological role of HLFMO3 and to determine the involvement of this enzyme in human disease states. Little is known about the substrate specificity of HLFMO3 and essentially nothing is known regarding the way HLFMO3 transforms endogenous amines to non-toxic readily excreted N-oxidized materials. HLFMO3 is a major component of the defense that protects humans against potentially toxic chemicals in their environment. Thus, humans with a decreased amount of HLFMO3 may be predisposed to the toxic or pathogenic action of xenobiotics and endogenous materials. The central hypothesis of our work is that human subjects with mutant HLFMO3 are predisposed to potentially dangerous disease states and the best method to study the effect of HLFMO3 structure on function is to begin parallel investigations of a mutant HLFMO3 identified from a human population. Because we have made significant progress in recent years towards developing several unique biochemical and molecular methods for the study of HLFMO3, it will be possible to rapidly determine a role of this enzyme in human drug and chemical biotransformations and human disease conditions. The proposal studies are divided into three major sections: 1) Confirm the identity and function of a region of mutant HLFMO3 involved in a human disease state, 2) Construct a HLFMO3 cDNA library and select and then express enzyme activity in E. coli, and 3) Examine the biochemical and physical properties of active cDNA-expressed HLFMO3 mutants.
The specific aims of section 1 include the verification of the sequence of adult HLFMO3 by comparing the structure of the isolated and purified wild-type enzyme with that of the mutant enzyme deduced from the cDNA isolated from human trimethylaminuria patients.
The specific aims of section 2 include construction of a cDNA library of 7 19 plasmic-encoded HLFMO3 genes (as well as the cDNA for the mutant(s) observed in trimethylaminuria patients), selection based on a novel screening procedure and expression of the active HLFMO3 mutants in E. coli.
The specific aims of section 3 include the verification of HLFMO3 enzyme activity utilizing rapid, efficient and highly sensitive HPLC methods for determination of endogenous and novel selective functional substrate activity. Comparison of the effects of altered amino acids on enzyme activity will be done with a determination of substrate free energy binding compared with wild-type enzyme. We will also specifically examine mutant HLFMO3 enzymes for enhanced thermostability because evolutionary selection for thermal stability may be almost as important as catalytic activity. The results of the present study will provide a detailed picture of the function of HLFMO3. The study will provide a basis for understanding a role of HLFMO3 in endogenous and xenobiotic human metabolism. The significance of the work is that it will lead to a more sophisticated understanding of a role of HLFMO3 in human disease and human health.
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