Pyrroloquinoline quinone biogenesis: characterization of PqqC and its H84N and H84A active site variants.
Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial vitamin that serves as a cofactor in numerous alcohol dehydrogenases. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF, and proceeds by an unknown pathway. The protein encoded by pqqC catalyzes the final step of PQQ formation, which involves a ring closure and an overall eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) in the absence of a redox-active metal or cofactor. A recent crystal structure has implicated numerous PQQ-PqqC interactions [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918]. To investigate the mechanism of the PqqC reaction, the active site residue His84 has been mutated to H84A and H84N, and the kinetic and spectroscopic properties have been compared to each other and the wild-type enzyme using aerobic and anaerobic conditions. Both mutants form PQQ under aerobic conditions with rate constants of 0.09 min-1 and 0.056 min-1 relative to 0.34 min-1 for the wild-type enzyme. In addition to the initial E-AHQQ complex (532-536 nm) and the product E-PQQ complex (346-366 nm), a number of spectral intermediates are observed between 316 and 344 nm. The anaerobic reaction is particularly informative, showing that while mixing of H84N with AHQQ leads to a 344 nm intermediate, this is unable to proceed to a final 318 nm species; by contrast H84A forms the 344 nm species as a precursor to the 318 nm species. In the context of the proposed chemical mechanism for PqqC [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918], we assign the 344 nm intermediate to a quinoid species and the 318 nm intermediate to an initial quinol species. The proposed role of H84 is as a proton donor to the oxyanion of the quinoid species such that subsequent C-H bond cleavage can occur to form a monoanionic quinol. In the absence of a proton donor (as occurs in H84N), the normal reaction path is precluded as this would require formation of an unstable, dianionic species. Unlike H84N, H84A appears to be small enough to allow entry of active site water, which is postulated to adopt the role of active site proton donor.
Magnusson
Olafur Th
OT
Department of Chemistry, University of California, Berkeley, California 94720, USA.
RoseFigura
Jordan M
JM
Toyama
Hirohide
H
Schwarzenbacher
Robert
R
Klinman
Judith P
JP
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D014644:Genetic Variation; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D045542:PQQ Cofactor / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C095467:PqqC protein, Bacteria; D011994:Recombinant Proteins; D045542:PQQ Cofactor