Functional and Evolutionary Relationships Among Diverse Oxygenases

Abstract

Oxygenases that incorporate one or two atoms of dioxygen into substrates are found in many metabolic pathways. In this article, representative oxygenases, principally those found in bacterial pathways for the degradation of hydrocarbons, are reviewed. Monooxygenases, discussed in this chapter, incorporate one hydroxyl group into substrates. In this reaction, two atoms of dioxygen are reduced to one hydroxyl group and one H2O molecule by the concomitant oxidation of NAD(P)H. Dioxygenases catalyze the incorporation of two atoms of dioxygen into substrates. Two types of dioxygenases, aromatic-ring dioxygenases and aromatic-ring-cleavage dioxygenases, are discussed. The aromatic-ring dioxygenases incorporate two hydroxyl groups into aromatic substrates, and cis-diols are formed. This reaction also requires NAD(P)H as an electron donor. Aromatic-ring-cleavage dioxygenases incorporate two atoms of dioxygen into aromatic substrates, and the aromatic ring is cleaved. This reaction does not require an external reductant. All the oxygenases possess a cofactor, a transition metal, flavin or pteridine, that interacts with dioxygen. The concerted reactions between dioxygen and carbon in organic compounds are spin forbidden. The cofactor is used to overcome this restriction. For the oxygenases that require the NAD(P)H cofactor, the enzyme reaction is separated into two steps, the oxidation of NAD(P)H to generate two reducing equivalents, and the hydroxylation of substrates. Flavoprotein hydroxylases that catalyze the monohydroxylation of the aromatic ring carry out these two reactions on a single polypeptide chain. In other oxygenases, the NAD(P)H oxidation and a hydroxylation reaction are catalyzed by two separate polypeptides that are linked by a short electron-transport chain. Two reducing equivalents generated by the oxidation of NAD(P)H are transferred through the electron-transport chain to the cofactor on a hydroxylase component that they reduce. Dioxygen couples with the reduced cofactor and subsequently hydroxylates substrates. The electron-transport chains associated with oxygenases contain at least two redox centers. The first redox center is usually a flavin, while the second is an iron-sulfur cluster. The electron transport is initiated by a single two-electron transfer from NAD(P)H to a flavin, followed by two single-electron transfers from the flavin to an iron-sulfur cluster. The primary sequences of many oxygenases have been determined, and according to their sequence similarities, the oxygenases can be grouped into several protein families. Among proteins of the same family, the sequences in regions involved in cofactor binding are strongly conserved. Local sequence similarities are also observed among oxygenases from different families, primarily in regions involved in cofactor binding.

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase ofEscherichia coliW: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

“…Oxygenases are the enzymes that catalyze the initial reactions of aerobic catabolic pathways for aromatic compounds by incorporating either two atoms of molecular oxygen (dioxygenases) or a single oxygen atom (monooxygenases) (14,15). For the monooxygenases that require the NAD(P)H cofactor, the reaction is separated into two steps, i.e., the oxidation of NAD(P)H to generate two reducing equivalents and the hydroxylation of substrates.…”

“…For the monooxygenases that require the NAD(P)H cofactor, the reaction is separated into two steps, i.e., the oxidation of NAD(P)H to generate two reducing equivalents and the hydroxylation of substrates. Most of the monooxygenases catalyzing the hydroxylation of the aromatic ring are flavoprotein enzymes that carry out the two reactions on a single polypeptide chain (14,15). However, multicomponent monoxygenases where NAD(P)H oxidation and the hydroxylation reaction are catalyzed by separate polypeptides linked by an electron transport chain have been also described (14,15).…”

“…Most of the monooxygenases catalyzing the hydroxylation of the aromatic ring are flavoprotein enzymes that carry out the two reactions on a single polypeptide chain (14,15). However, multicomponent monoxygenases where NAD(P)H oxidation and the hydroxylation reaction are catalyzed by separate polypeptides linked by an electron transport chain have been also described (14,15). The most complex monooxygenases described so far are the six-component proteins for the hydroxylation of aromatic compounds, such as phenol, benzene, and toluene (5,14,15).…”

“…Within the heme monooxygenase group, a flavoprotein constitutes the electron transfer component, and cytochrome c or cytochrome P450 is usually found as the oxygenase component (14,15). The nonheme two-component monooxygenases can use either pteridines or flavins as cofactors (15).…”

Evolutionary relationships among extradiol dioxygenases

“…In contrast, extradiol dioxygenases cleave meta to the hydroxyl substituents and typically depend on nonheme Fe(II). Although these distinctions may appear to be minor, they are in fact a manifestation of enzymes that have completely different structures and exclusively utilize different mechanisms (18).…”