Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Abstract

The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin-and flavodoxin reductasebased reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.Pyrroloquinoline quinone (PQQ) 2 is employed by a wide variety of bacteria, where it functions as a redox cofactor in substrate oxidation via an alternate (non-glycolytic) pathway for production of cellular ATP (1). Hundreds of bacterial species are thought to produce PQQ, based on the presence in their genomes of the strongly conserved pqq operon (2) (Fig. 1A). Although the existence of this important redox cofactor has been known for decades, knowledge of the biosynthetic pathway for PQQ has remained scant (3, 4). The PQQ molecule derives from the evolutionarily conserved glutamate and tyrosine side chains within a ribosomally produced peptide substrate PqqA (Fig. 1, B and C). To produce PQQ, a large number of chemical modifications are required: (i) the formation of a new carbon-carbon bond between the ␥-carbon of glutamate and the 3-position of tyrosine (labeled in green in Fig. 1C); (ii) the addition of two additional hydroxyl groups to the tyrosine ring; (iii) a condensation between the 4-OH of tyrosine and the backbone amine of glutamate to produce a heterocyclic ring; and (iv) an 8-electron oxidation catalyzed by PqqC, a cofactorless enzyme that converts 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) to PQQ in the final step of the pathway (5-7).The radical SAM enzyme PqqE has been the primary candidate as the catalyst for the formation of the new carbon-carbon bond between glutamate and tyrosine. Radical SAM proteins use the reductive cleavage of S-adenosyl methionine to initiate free radical chemistry, and can accomplish a wide variety of reactions, including carbon-carbon bond formation (8, 9). PqqE is a founding member of the SPASM domain-containing radical SAM proteins, which contain one or more auxiliary clusters in their C-terminal regions, and of which several are known to modify small peptides or proteins (10, 11). Recently, a small (ϳ10 kDa) protein, PqqD, has been shown to form a strong, sub-micromolar K D complex with the peptide substrate PqqA. This complex was further demonstrated to associate with PqqE (12). These findings suggested that PqqD would serve as a chaperone to deliver PqqA to PqqE, functioning as a necessary and heretofore missing componen...

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

“…This study provided new insights regarding the role of PqqD 5 and clarified several questions regarding PQQ biosynthesis. 71 Further studies will be required to delineate the role of the auxiliary clusters and the precise details of the crosslinking reaction.…”

Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA

“…PqqD has also been recently shown to be necessary for the function of radical S -adenosylmethionine protein PqqE. 19 However, no structure of the PqqA/PqqD complex is currently available.…”

Lessons From the Studies of a C C Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis

“…In general, many radical SAM enzymes catalyze the abortive cleavage of SAM. In some cases, the abortive cleavage is caused by the inappropriate position of affinity tag (Grove, Ahlum, Sharma, Krebs, & Booker, 2010), or the use of non-physiological substrate (Yang et al, 2012), or reductant (Barr et al, 2016). Therefore, the extent of the abortive cleavage can be used for quality control of radical SAM enzyme preparation.…”