PqqB (VPARA_00950, A0A0H2M8Q2; also annotated as pqqB1) in Variovorax paradoxus is a 307-amino-acid metallo-beta-lactamase-fold non-heme iron-dependent hydroxylase (HAMAP MF_00653, InterPro IPR011842, NCBIfam TIGR02108, PANTHER PTHR42663:SF7) encoded in the pqqBCDE (VPARA_00950-VPARA_00920) pyrroloquinoline quinone (PQQ) biosynthetic gene cluster. Although older automated rules annotated PqqB as a putative periplasmic PQQ transporter, biochemical and crystallographic characterization of bacterial PqqB orthologs established that PqqB is a cytosolic Fe(2+)-dependent oxygenase/hydroxylase that catalyzes oxygen insertion into the tyrosine ring of a PqqA-derived Glu-Tyr cross-linked intermediate to generate the hydroxyquinone precursor that cyclizes into AHQQ prior to terminal oxidation by PqqC.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0018189 pyrroloquinoline quinone biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Reason: PqqB (VPARA_00950, A0A0H2M8Q2) is an essential component of the conserved pqqBCDE gene cluster in Variovorax paradoxus and catalyzes the iron-dependent hydroxylation steps of pyrroloquinoline quinone biosynthesis (PMID:30811189, PMID:32731194). Supporting Evidence: UniProtKB:A0A0H2M8Q2 CC -!- PATHWAY: Cofactor biosynthesis; pyrroloquinoline quinone biosynthesis. CC {ECO:0000256|ARBA:ARBA00004886, ECO:0000256|HAMAP-Rule:MF_00653}. PMID:30811189 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. |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | ISS PMID:30811189 Discovery of Hydroxylase Activity for PqqB Provides a Missin... | NEW | Reason: Biochemical and 18O2-labeling studies of orthologous bacterial PqqB (PMID:30811189, PMID:32731194) demonstrate that PqqB is a non-heme Fe(2+)-dependent hydroxylase that incorporates oxygen from O2 into the aromatic ring of a PqqA-derived cross-linked intermediate during PQQ biosynthesis; GO:0016705 is the appropriate molecular function term pending creation of a PqqB-specific hydroxylase term (matching PSEPK/pqqB and MODULE:pqq_biosynthesis). Supporting Evidence: PMID:30811189 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. PMID:32731194 Detailed kinetic analyses have confirmed that PqqB is a previously unknown iron-dependent hydroxylase, indicating a cross-over between the MBL superfamily and the non-heme iron hydroxylases. |
| GO:0005506 iron ion binding | ISS PMID:30811189 Discovery of Hydroxylase Activity for PqqB Provides a Missin... | NEW | Reason: PqqB binds a catalytic Fe(2+) ion at its conserved metallo-beta-lactamase active site (2-His/1-Asp motif) to catalyze O2-dependent aromatic hydroxylation during PQQ biosynthesis (PMID:30811189, PMID:32731194). Supporting Evidence: PMID:30811189 Only a single metal, Fe2+, is observed to undergo increase in affinity to Me PqqB in the presence of both Cys-Tyr (ca. 3-fold) and Cys-DOPA (ca. 10-fold), Fig. 2b. |
| GO:0005829 cytosol | IC UniProtKB:A0A0H2M8Q2 | NEW | Reason: Inferred by curator (IC) because V. paradoxus PqqB (A0A0H2M8Q2) is a soluble 307-residue enzyme lacking signal peptides or transmembrane helices that acts intracellularly on cytosolic PqqA-derived intermediates upstream of cytosolic PqqC. Supporting Evidence: UniProtKB:A0A0H2M8Q2 FT DOMAIN 53..274 FT /note="Metallo-beta-lactamase" FT /evidence="ECO:0000259|Pfam:PF12706" |
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Download this section (compressed HTML)Q: Does Variovorax paradoxus PqqB (VPARA_00950) act on the excised Glu-Tyr cross-linked dipeptide or on a partially proteolyzed PqqA peptide intermediate in vivo, and what is the role of 2-oxoglutarate in the first hydroxylation step?
Experiment: Express and purify recombinant Variovorax paradoxus PqqB (A0A0H2M8Q2), reconstitute with Fe(2+) and Zn(2+), and assay O2-dependent hydroxylation of synthetic Glu-Tyr and Cys-DOPA cross-linked dipeptide substrates in the presence and absence of 2-oxoglutarate by LC-HRMS and 18O2 isotopic labeling.
Hypothesis: Fe(2+)-reconstituted V. paradoxus PqqB catalyzes O2-dependent aromatic ring hydroxylation of the cross-linked tyrosine moiety to generate the trihydroxyquinone precursor of AHQQ.
Type: In vitro metalloenzyme reconstitution and 18O2 LC-HRMS assay
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