Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution.
p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000. It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller. Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third. An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives. The phases were improved with the B.C. Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis. The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system. The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system. The flavoprotein subunit consists of three domains in close contact. The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site. The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side. The flavin ring lies at the juncture of the first two domains. The third domain lies against the first domain and helps cover the rest of the FAD chain. The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit. The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart. The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.
Mathews
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FS
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Chen
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ZW
Bellamy
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HD
McIntire
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WS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013816:Thermodynamics; D014961:X-Ray Diffraction / Q000379:methods; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D020245:p-Chloromercuribenzoic Acid; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C019947:p-cresol oxidoreductase; C064500:flavocytochrome c sulfide dehydrogenase