The covalent structure of the small subunit from Pseudomonas putida amine dehydrogenase reveals the presence of three novel types of internal cross-linkages, all involving cysteine in a thioether bond.
Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the beta-carbon atom of an aspartic acid, one with the gamma-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.
Vandenberghe
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Laboratorium voor Eiwitbiochemie en Eiwitengineering, K.L. Ledeganckstraat, 35, 9000 Gent, Belgium.
Kim
J K
JK
Devreese
B
B
Hacisalihoglu
A
A
Iwabuki
H
H
Okajima
T
T
Kuroda
S
S
Adachi
O
O
Jongejan
J A
JA
Duine
J A
JA
Tanizawa
K
K
Van Beeumen
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D045563:Indolequinones; D013058:Mass Spectrometry; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D016958:Pseudomonas putida / Q000201:enzymology*; D011809:Quinones / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013440:Sulfides / Q000737:chemistry*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014965:X-Rays
D000596:Amino Acids; D003432:Cross-Linking Reagents; D045563:Indolequinones; D010455:Peptides; D011809:Quinones; D013440:Sulfides; C070569:tryptophan tryptophylquinone; D018698:Glutamic Acid; D006418:Heme; D014364:Tryptophan; D000587:Oxidoreductases Acting on CH-NH Group Donors; C119497:quinohemoprotein amine dehydrogenase; D003545:Cysteine