Biosynthesis of Topa Quinone Cofactor in Bacterial Amine Oxidases

Abstract

Resonance Raman spectroscopy is an excellent technique for providing structural information on the 2,4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper-containing amine oxidases. This technique has been used to investigate the copper- and O2-dependent biosynthesis of the TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxidase from Arthrobacter globiformis. Incubation of the holoenzyme in H218O causes frequency shifts at 1684(-26) cm-1 in PEAO and at 1679(-28) cm-1 in histamine oxidase, allowing this feature to be assigned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O2 in the presence of H218O, the resultant holoproteins show increased shifts of -3 to -6 cm-1 in a number of other vibrational modes, particularly at 411 and 1397 cm-1. Because these small shifts persist when the H218O-regenerated protein is back-exchanged into H216O, they can be assigned to oxygen isotope substitution at the C-2 postion. No isotope shifts are observed when apoprotein is regenerated with Cu(II) in the presence of 18O2. Thus, it is concluded that the C-2 oxygen atom of TPQ originates from H2O rather than O2. The isotope dependence of the 1397-cm-1 mode allows it to be assigned to the C O moiety at the C-2 position, with its low frequency being indicative of only partial double bond character. Similar frequency shifts due to 18O at C-2 are observed in the resonance Raman spectra of H218O-regenerated PEAO after derivatization of the C-5 carbonyl with either p-nitrophenylhydrazine (-5 cm-1 at 480 cm-1) or methylamine (-5 cm-1 at 1301 cm-1). Taken together, these results indicate that the TPQ cofactor in the native enzyme has substantial electron delocalization between the C-2 and C-4 oxygens and that only the C-5 oxygen has predominantly C=O character.

Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis

“…TPQ biogenesis is a Cu II -dependent process that consumes two molecules of O 2 and involves the insertion of two oxygen atoms into the phenolic ring of an active site tyrosine (Scheme B). − Although isotopic labeling studies (to determine whether the TPQ oxygens derive from O 2 or solvent) are not feasible due to rapid exchange, crystallographic, mutant, and model studies have shown that the AO biogenesis reaction occurs through one monooxygenation and one hydration step (Scheme B). However, little is known about the mechanistic nature of these steps or the geometric and electronic structure adopted by the active site copper prior to or during the biogenesis reaction.…”

Human copper-dependent amine oxidases

“…Dopaquinone (DPQ) is proposed to be the common intermediate during the biogenesis of TPQ and LTQ, where the 1,4-addition of either water or the ε-amino side chain of a peptidyl lysine residue to DPQ yields TPQ or LTQ, respectively (13,14) (Figure 2). A careful inspection of the reaction product of TPQ biogenesis in the presence of H 2 18 O and 18 O 2 by resonance Raman spectroscopy revealed that the C2 oxygen of TPQ is from solvent water, rather than O 2 (17). In the same study, substantial electron delocalization between the C2 and C4 oxygens of the TPQ cofactor was observed, whereas the C5=O bond had more carbonyl character.…”

Mutation of a Strictly Conserved, Active-Site Residue Alters Substrate Specificity and Cofactor Biogenesis in a Copper Amine Oxidase

“…These components, in the absence of other factors, including reducing equivalents, are sufficient for the conversion of tyrosine to TPQ in Vitro (6). Mechanisms involving a copper hydroperoxide (7) or tyrosyl radical (8) have been put forth; however, neither mechanism has been supported by experimental evidence nor is there a clear understanding as to which residues may be critical for biogenesis to occur.…”