Active Site Aspartate Residues Are Critical for Tryptophan Tryptophylquinone Biogenesis in Methylamine Dehydrogenase

Abstract

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue ␤Trp 57 and covalent cross-linking of ␤Trp 57 to ␤Trp 108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues ␤Asp 76 and ␤Asp 32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative ␤D76N and ␤D32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. ␤D76N MADH possessed the six disulfides but had no oxygen incorporated into ␤Trp 57 and was completely inactive. The ␤D32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into ␤Trp 57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the ␤D32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the K m value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.Protein-derived cofactors are catalytic and redox-active prosthetic groups that are derived from posttranslational modification of peptide amino acid residues (1-3). These include covalently cross-linked amino acid residues, stable free radicals derived from amino acid residues, and quinones derived from tyrosine and tryptophan residues. The protein-derived cofactor tryptophan tryptophylquinone (TTQ) 1 (4) is the prosthetic group of methylamine dehydrogenase (MADH) (Fig. 1) (5). MADH is a periplasmic enzyme that catalyzes the oxidative deamination of methylamine to formaldehyde plus ammonia and the subsequent electron transfer to a type I copper protein, amicyanin (6). MADH from Paracoccus denitrificans is composed of four subunits that are arranged in a symmetrical ␣ 2 ␤ 2 structure (7). A TTQ is located in each ␤ subunit and is formed by posttranslational modifications of ␤Trp 57 and ␤Trp 108 . As can be seen from its structure (Fig. 1), TTQ biosynthesis requires insertion of oxygens at the C-6 and C-7 positions of the indole ring of ␤Trp 57 and formation of a covalent bond between the indole rings of ␤Trp 57 and ␤Trp 108 . TTQ has also been identified as the prosthetic group of aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis (8). As in MADH, the TTQ is located in the smaller ␤ subunit of AADH, which is 60% identical to the MADH ␤ subunit (9). In addition to TTQ, the MADH ␤ subunit exhibits another unusual structural feature. Of the 131 amino acid residues of the ␤ subunit, there are 12 cysteines, all of which are conserved in MADH from other organisms and in AADH and which form six intrasubunit disulfide bonds (7)...

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

“…Furthermore, it has been reported that mutation of the non-oxygenated TTQ partner, ␤Trp-108, into Cys leads to the generation of CTQ instead of TTQ in MADH (26), strongly suggesting that biogenesis of TTQ and CTQ shares the common mechanisms at least partly, proceeding within the periplasm. Finally, it is also interesting to note that the mauD gene required for MADH maturation codes for a protein similar to disulfide isomerase that may be involved in the periplasmic processing of the six disulfide bonds of the ␤-subunit of MADH (38), which likely provides a proper positioning of active-site residues involved in TTQ biogenesis (29). Although the ␥-subunit of QHNDH does not contain disulfide bonds, it does contain three Cysto-Asp/Glu thioether cross-links that appear functionally equivalent to disulfide bonds.…”

Roles of active site residues in LodA, a cysteine tryptophylquinone dependent ε-lysine oxidase

“…A similar effect was reported when the structurally analogous Asp was mutated in methylamine dehydrogenase (MADH). That led to expression of a precursor of MADH without TTQ [10]. …”

“…Two other quinoproteins, the CTQ-containing quinohemoprotein amine dehydrogenase (QHNDH) [12] and the TTQ-containing MADH [13], have a structurally conserved Asp residue in the position corresponding to Cys448 in the LodA structure in the active site. Mutation of the corresponding Asp in MADH affected the efficiency of MauG-dependent TTQ biosynthesis but had no effect on the catalytic activity of the population of the isolated MADH with fully formed TTQ [10]. As such, Cys448 in LodA was converted to Asp, as well as Ala, to ascertain its role in CTQ biosynthesis or catalysis or both.…”

The Silent Form of Quinohemoprotein Amine Dehydrogenase fromParacoccus denitrificans

“…[12][13][14] According to CTQ, putative iron sulfur cluster binding protein probably belongs to the Radical SAM superfamily and can be involved in the biogenesis of QHAmDH, 11) but the details remain obscure. If sQHAmDH is the precursor of QH-AmDH, sQH-AmDH might become a key protein in understanding the biogenesis of post-transcriptionally derived quinone cofactors.…”

“…11) In TTQ biosynthesis, MauG protein, an oxygen-binding diheme c protein, would be involved in cross-linking reaction of the two tryptophans. [12][13][14] The largest subunit of QH-AmDH is a 489-residue, a four-domain polypeptide chain that contains two heme c cofactors. Heme c(a) is solvent-accessible and has His and Met as axial ligands.…”