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Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity.

ABSTRACT: The flavin-dependent amine oxidase (FAO) superfamily consists of over 9000 nonredundant sequences represented in all domains of life. Of the thousands of members identified, only 214 have been functionally annotated to date, and 40 unique structures are represented in the Protein Data Bank. The few functionally characterized members share a catalytic mechanism involving the oxidation of an amine substrate through transfer of a hydride to the FAD cofactor, with differences observed in substrate specificities. Previous studies have focused on comparing a subset of superfamily members. Here, we present a comprehensive analysis of the FAO superfamily based on reaction mechanism and substrate recognition. Using a dataset of 9192 sequences, a sequence similarity network, and subsequently, a genome neighborhood network were constructed, organizing the superfamily into eight subgroups that accord with substrate type. Likewise, through phylogenetic analysis, the evolutionary relationship of subgroups was determined, delineating the divergence between enzymes based on organism, substrate, and mechanism. In addition, using sequences and atomic coordinates of 22 structures from the Protein Data Bank to perform sequence and structural alignments, active-site elements were identified, showing divergence from the canonical aromatic-cage residues to accommodate large substrates. These specificity determinants are held in a structural framework comprising a core domain catalyzing the oxidation of amines with an auxiliary domain for substrate recognition. Overall, analysis of the FAO superfamily reveals a modular fold with cofactor and substrate-binding domains allowing for diversity of recognition via insertion/deletions. This flexibility allows facile evolution of new activities, as shown by reinvention of function between subfamilies.

SUBMITTER: Tararina MA

PROVIDER: S-EPMC7321841 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s(-1). However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s(-1) at air saturation) and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While β-mercaptoethanol is a very poor substrate of sfALR (∼0.3 min(-1) at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be physiologically important in preventing the oxidase from catalyzing the potentially harmful oxidation of intracellular glutathione.

Dataset Information

Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity.

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