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 reductase-based 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.

Keywords: PQQ; S-adenosylmethionine (SAM); SPASM domain; flavoprotein; iron-sulfur protein; peptides; post-translational modification (PTM); radical SAM.

FIGURE 1.

A, the pqq operon contains, at a minimum, the genes PqqA, PqqB, PqqC, PqqD, and PqqE. PqqF, a protease, is often present as well, but is not uniformly conserved. The gene order is strictly conserved. In M. extorquens AM1, there is a gene fusion between PqqC and PqqD. B, alignment of PqqA from PQQ producing organisms. Asterisks denote conserved residues. The conserved glutamate and tyrosine near the C terminus are modified to form PQQ. C, PQQ, showing the origin of the atoms that make its backbone. The bond highlighted in green is the carbon-carbon bond hypothesized to be made by PqqE.

FIGURE 2.

Reconstitution of M. extorquens PqqE shows a 4Fe-4S protein. A, zero-field Mössbauer spectrum of PqqE at 4 K, as recombinantly expressed in the presence of ⁵⁷Fe and purified from E. coli. Black circles show the raw data, which have been offset from the axis by 0.5% for ease of interpretation. These data are decomposed into 4Fe-4S (solid line) and 2Fe-2S (dotted line) components. B, Mössbauer spectrum of as-isolated PqqE reconstituted with additional ⁵⁷Fe and sulfide; the 4Fe-4S component comprises 80% of all iron in the sample. C, absorbance (Abs.) spectrum of PqqE before (dashed lines) and after (solid lines) reconstitution of the as-isolated protein.

FIGURE 3.

Modification of PqqA by PqqE and PqqD monitored by LC-MS. A, the initial 2+ ion mass envelope of unmodified PqqA. B, the 2+ mass envelope of a minor peak, eluting 1 min earlier, seen following a 24-h reaction under anaerobic conditions. A noticeable shift in mass by 2 Da is observed, consistent with cross-linking of residues in PqqA according to Fig. 5. In both A and B, the red spectra are calculated mass envelopes for PqqA (A) and modified PqqA (B), offset slightly for ease of comparison. Arrows indicate the most abundant ions, which were used to quantify the relative amount of modified PqqA. C, chromatograph showing the elution profile of 1537.7 (black) and 1536.7 (blue) ions in an unreacted PqqA sample. D, chromatograph showing the elution profile of 1537.7 (black) and 1536.7 (blue) ions in a 24-h reaction mixture; a small peak containing cross-linked PqqA is seen to elute earlier than the unreacted PqqA.

FIGURE 4.

MS-MS analysis of modified (top) and unmodified (bottom) PqqA. The modified peptide shows cleavage protection between E and Y; few or none of the fragments detected in the parent PqqA are observed. Mass fragments are labeled according to the sequence of PqqA shown in the top panel. b ions that are not present in the cross-linked sample are outlined in the dashed box.

FIGURE 5.

Scheme for the formation of PQQ from glutamate and tyrosine of PqqA. PqqA's conserved glutamate undergoes a hydrogen abstraction on the γ-carbon, forming a carbon radical that reacts with the 3-position of tyrosine to form a carbon-carbon bond. At this point, re-aromatization is favored, and the modified tyrosine loses a proton and electron, potentially to either the radical SAM cluster or an auxiliary cluster.