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

Abstract

Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB-E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ␣-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K D of 300 nM. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.

Gene products and processes contributing to lanthanide homeostasis and methanol metabolism in Methylorubrum extorquens AM1

“…An aminopeptidase was also identified which may be involved in processing one or more proteins required for XoxF1 and MxaFI function. Alternatively, the aminopeptidase could function in processing PQQ, as PQQ is peptide based and not all PQQ processing proteins have been identified 53 , 54 .…”

New developments in RiPP discovery, enzymology and engineering

“…254,255 A two-component heterodimeric protease PqqFG from Methylobacterium extorquens AM1, a protein that is not always encoded in the PQQ BGC, was able to hydrolyze linear PqqA, preferentially hydrolyzing the peptide both N-and C-terminal to Ser residues. 256 Previously, a PqqF homolog from Serratia sp. FS14 had been crystallized demonstrating an HxxEH Zn-binding motif.…”

“…The unstructured substrate peptide is fed through a narrow channel and is progressively hydrolyzed until sufficient steric hindrance prevents further processing. A similar heterodimeric zinc-dependent protease was also implicated in the biosynthesis of PQQ (Section 3.21) 256,257 and some pearlins (Section 3.24). 277 4.4.4 AplP-like proteases.…”

Correlational networking guides the discovery of cryptic natural product biosynthetic enzymes

“…As expected, in vitro enzymatic assays using conditions described above revealed corresponding products cleaved from the conserved Q-A-(A/V)-(D/E) motif of PttA2-PttA7 (Supplementary Figs. [33][34][35][36]. The products also showed different overhangs of amino acids at the N-terminus (Supplementary Figs.…”

“…In addition, formation of these products was also dependent on the precursor modification by PttKC (Supplementary Figs. [33][34][35][36].…”