Further Insights into Quinone Cofactor Biogenesis:  Probing the Role of mauG in Methylamine Dehydrogenase Tryptophan Tryptophylquinone Formation

• Published in: Biochemistry (Easton) 2004-05, Vol.43 (18), p.5494-5502
• Main Authors: Pearson, Arwen R, De la Mora-Rey, Teresa, Graichen, M. Elizabeth, Wang, Yongting, Jones, Limei H, Marimanikkupam, Sudha, Agger, Sean A, Grimsrud, Paul A, Davidson, Victor L, Wilmot, Carrie M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chymotrypsin - chemistry; Coenzymes - biosynthesis; Cytochrome-c Peroxidase - chemistry; Cytochrome-c Peroxidase - genetics; Electrophoresis, Polyacrylamide Gel; Histidine - genetics; Hydrolysis; Indolequinones - biosynthesis; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidoreductases Acting on CH-NH Group Donors - chemistry; Oxidoreductases Acting on CH-NH Group Donors - genetics; Oxidoreductases Acting on CH-NH Group Donors - metabolism; Paracoccus denitrificans - enzymology; Paracoccus denitrificans - genetics; Protein Processing, Post-Translational; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin - chemistry; Tryptophan - analogs & derivatives; Tryptophan - biosynthesis; Valine - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi049863l

Paracoccus denitrificans methylamine dehydrogenase (MADH) is an enzyme containing a quinone cofactor tryptophan tryptophylquinone (TTQ) derived from two tryptophan residues (βTrp57 and βTrp108) within the polypeptide chain. During cofactor formation, the two tryptophan residues become covalently linked, and two carbonyl oxygens are added to the indole ring of βTrp57. Expression of active MADH from P. denitrificans requires four other genes in addition to those that encode the polypeptides of the MADH α2β2 heterotetramer. One of these, mauG, has been shown to be involved in TTQ biogenesis. It contains two covalently attached c-type hemes but exhibits unusual properties compared to c-type cytochromes and diheme cytochrome c peroxidases, to which it has some sequence similarity. To test the role that MauG may play in TTQ maturation, the predicted proximal histidine to each heme (His35 and His205) has each been mutated to valine, and wild-type MADH was expressed in the background of these two mauG mutants. The resultant MADH has been characterized by mass spectrometry and electrophoretic and kinetic analyses. The majority species is a TTQ biogenesis intermediate containing a monohydroxylated βTrp57, suggesting that this is the natural substrate for MauG. Previous work has shown that MADH mutated at the βTrp108 position (the non-oxygenated TTQ partner) is predominantly also this intermediate, and work on these mutants is extended and compared to the MADH expressed in the background of the histidine to valine mauG mutations. In this study, it is unequivocally demonstrated that MauG is required to initiate the formation of the TTQ cross-link, the conversion of a single hydroxyl located on βTrp57 to a carbonyl, and the incorporation of the second oxygen into the TTQ ring to complete TTQ biogenesis. The properties of MauG, which are atypical of c-type cytochromes, are discussed in the context of these final stages of TTQ biogenesis.