We will continue our studies of the biosynthesis, 3-dimensional structure, and chemical and election transfer mechanisms for p-cresol methylhydroxylase (PCMH) from Pseudomonas putida. The 2.5 A structure of this alpha2beta2 flavocytochrome is known, as is the 3.o A structure of the PCMH/p-cresol complex, and the structural genes for the flavoprotein and cytochrome subunits have been cloned and sequenced. The normal cytochrome subunit is expressed in E. coli, however, when the flavoprotein is expressed in this organism, in the absence of the cytochrome, FAD is noncovalently- bound, although the flavoprotein is active. When the cytochrome subunit is added to the flavoprotein, the FAD becomes covalently-bound, as in PCMH made by P. putida. We plan to study the covalent flavination in more detail, and take advantage of this phenomenon to reconstitute the flavoprotein with FAD analogues. This should provide important information concerning the function of this enzyme. Based on the crystal structure, we will create site-specifically-mutated forms of the flavoprotein and cytochrome subunits to further study the covalent flavination, the chemical mechanism, subunit interaction and electron transfer from FAD to heme. Experiments are planned to investigate the interaction of this enzyme with horse heart cytochrome c, and protein electron acceptor(s) from P. putida. X-ray crystallographic analysis of P. putida- and E. coli-expressed PCMH will continue. We will continue studies of the 3-dimensional structures of the enzyme various oxidation states, and int he presence of substrate analogues. Additionally, we will attempt to obtain the x-ray structures of the unassociated subunits. The NMR study of the solution structure of the cytochrome subunit will be continued.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
2P01HL016251-21A1
Application #
3736003
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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