Discovery of Hydroxylase Activity for PqqB Provides a Missing Link in the Pyrroloquinoline Quinone Biosynthetic Pathway

Abstract

Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ) which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo β-lactamase protein family and expand the catalytic repertoire of non-heme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

Phenylalanine meta‐Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation

“…A radical abstraction followed by oxygen rebound, akin to α‐ketoglutarate‐dependent dioxygenases, would result in loss of the ipso ‐deuterium. Although a radical mechanism has recently been proposed for the Fe II dependent hydroxylase PqqB as a key step in the biosynthesis of the cofactor pyrroloquinoline quinone (PQQ), this enzyme does not use a pterin cofactor; rather, dioxygen is directly activated by the Fe II centre . To date, there is no evidence to support a radical mechanism for iron‐ and pterin‐dependent hydroxylases .…”

Biological Pincer Complexes

“…An Fe-dependent hydroxylase, PqqB, converts the modified tyrosine into a hydroxyquinone (Scheme 1). [22] The structure of PqqB from Pseudomonas putida KT2440 was determined (PDB ID 6E13) and found to have a metallo β-lactase fold with two metal-binding sites per monomer; one site binds a structural zinc atom and the second binds iron using an aspartate and two histidine residues. The structure has zinc substituted for the catalytic iron atom and has the bound substrate analog 5cysteinyl-3,4-dihydroxyphenylalanine.…”

“…Two potential mechanisms of the enzyme were proposed, but further studies are needed to establish the steps of this chemical transformation. [22] The product of PqqB, 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ), is converted to PQQ by PqqC, a seven-helix bundle protein (PDB ID 1OTW), that catalyzes an 8-electron oxidation without the use of an enzyme cofactor. [23] Three of these oxidation steps require dioxygen and produce hydrogen peroxide, but one oxidative reaction uses hydrogen peroxide that is reduced to two molecules of water (Scheme 1).…”

A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone

“…Isolation by affinity (23,38) and PqqC (25). The reaction catalyzed by PqqB has been inferred using substrate analogs (24).…”

“…The biosynthesis of PQQ is initiated by the formation of a C-C bond between glutamate and tyrosine residues in PqqA, with the help of the chaperone PqqD, creating a cross-linked product PqqA* (23). The steps that follow this reaction include the processing of PqqA* by a protease and the action of PqqB, which was recently found to be an iron-dependent hydroxylase that installs the quinone moiety of PQQforming AHQQ (24). The last reaction of the pathway is catalyzed by PqqC, the first enzyme to be characterized in this pathway (25)(26)(27).…”