Article
Biochemistry, Vol. 50, No. 9, 2011 1565
The challenge for the future will be to tease apart the detailed
properties of the O core that contribute to O binding versus
(PQQ): implications for its function in biological systems. Biochem.
Pharmacol. 65, 67–74.
. Pearson, A. R., Jones, L. H., Higgins, L. A., Ashcroft, A. E., Wilmot,
C. M., and Davidson, V. L. (2003) Understanding quinone cofactor
biogenesis in methylamine dehydrogenase through novel cofactor
generation. Biochemistry 42, 3224–3230.
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0. Toyama, H., Chistoserdova, L., and Lidstrom, M. E. (1997) Sequence
analysis of pqq genes required for biosynthesis of pyrroloquinoline
quinone in Methylobacterium extorquens AM1 and the purification of
a biosynthetic intermediate. Microbiology 143, 595–602.
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catalysis. We consider it important that addition of O to mutant
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proteins promotes the appearance of new spectral species. This
is the second example of a special role for nonmetal O binding
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in promoting a conformational rearrangement during quino-
cofactor biogenesis. In an earlier study of TPQ formation, it was
shown that a charge transfer complex between the active site
copper and the tyrosine (Y405) that is the precursor to TPQ occurs
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only after exposure of anaerobic protein samples to O (40, 41). It
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appears that for both TPQ and PQQ biogenesis O binding can
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contribute to a restructuring of the active site that alters proton
movement involving deprotonation (of Y405) in the case of TPQ
biogenesis and most likely protonation of the quinoid intermedi-
ate in PQQ formation catalyzed by PqqC.
11. Houck, D. R., Hanners, J. L., and Unkefer, C. J. (1991) Biosynthesis
of pyrroloquinoline quinone. 2. Biosynthetic assembly from gluta-
mate and tyrosine. J. Am. Chem. Soc. 113, 3162–3166.
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2. Magnusson, O. T., Toyama, H., Saeki, M., Rojas, A., Reed, J. C.,
Liddington, R. C., Klinman, J. P., and Schwarzenbacher, R. (2004)
Quinone biogenesis: structure and mechanism of PqqC, the final
catalyst in the production of pyrroloquinoline quinone. Proc. Natl.
Acad. Sci. U.S.A. 101, 7913–7918.
CONCLUSION
The aggregate data presented in this study are consistent with
13. Magnusson, O. T., Toyama, H., Saeki, M., Schwarzenbacher, R., and
Klinman, J. P. (2004) The structure of a biosynthetic intermediate of
pyrroloquinoline quinone (PQQ) and elucidation of the final step of
PQQ biosynthesis. J. Am. Chem. Soc. 126, 5342–5343.
14. Puehringer, S., RoseFigura, J. M., Metlitzky, M., Toyama, H., Klin-
man, J. P., and Schwarzenbacher, R. (2010) Structural studies of
mutant forms of the PQQ-forming enzyme PqqC in the presence of
product and substrate. Proteins: Struct., Funct., Bioinf. 78, 2554–2562.
an “O core” in PqqC. We have characterized four mutations that
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implicate the vital roles played by H154, Y175, and R179, with a
change at any single position inhibiting PQQ production via a
possible impact on a protein conformation change to the closed
structure (Y175S) or a direct impairment of oxidative chemistry
(
H154N, Y175F, R179S). In the latter cases, it is further
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5. Magnusson, O. T., RoseFigura, J. M., Toyama, H., Schwarzenbacher,
R., and Klinman, J. P. (2007) Pyrroloquinoline quinone biogenesis:
characterization of PqqC and its H84N and H84A active site variants.
Biochemistry 46, 7174–7186.
6. Schwarzenbacher, R., Stenner-Liewen, F., Liewen, H., Reed, J. C.,
and Liddington, R. C. (2004) Crystal structure of PqqC from Kleb-
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siella pneumoniae at 2.1 A resolution. Proteins: Struct., Funct., Bioinf.
proposed that occupancy at the O binding pocket respositions
H84, a residue previously shown (15) to be a requisite proton
donor in quinol formation. Although the precise chemical nature
of the species formed from the single turnover of “O core”
mutants is not yet demonstrated, it is clear that these can persist
for long times in the presence of O . The present results, showing
a binding of O to mutant forms of PqqC, add another example
to the literature of gas binding pockets that can be generated in
the absence of either an organic cofactor or metal ion.
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56, 401–403.
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7. Meulenberg, J. J. M., Sellink, E., Loenen, W. A. M., Riegman, N. H.,
Vankleef, M., and Postma, P. W. (1990) Cloning of Klebsiella
pneumoniae PQQ genes and PQQ biosynthesis in Escherichia coli.
FEMS Microbiol. Lett. 71, 337–344.
8. Meulenberg, J. J. M., Sellink, E., Riegman, N. H., and Postma, P. W.
(1992) Nucleotide-sequencing and structure of the Klebsiella pneumo-
niae PQQ operon. Mol. Gen. Genet. 232, 284–294.
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ACKNOWLEDGMENT
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9. Kriss, G. A. (1994) Astronomical Data Analysis Software & Systems III.
0. Olsthoorn, A. J. J., and Duine, J. A. (1996) Production, characteriza-
tion, and reconstitution of recombinant quinoprotein glucose dehy-
drogenase (soluble type; EC 1.1.99.17) apoenzyme of Acinetobacter
calcoaceticus. Arch. Biochem. Biophys. 336, 42–48.
We thank Dr. Florence Bonnot for assistance with the PQQH2
reoxidation analysis.
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1. Nabiullin, A. A., Fedoreev, S. A., and Deshko, T. N. (1984) Circular
dichroism of quinoid pigments from Far Eastern representatives of
the family Boraginaceae. Chem. Nat. Compd. 19, 532–537.
2. Toyama, H., Fukumoto, H., Saeki, M., Matsushita, K., Adachi, O.,
and Lidstrom, M. E. (2002) PqqC/D, which converts a biosynthetic
intermediate to pyrroloquinoline quinone. Biochem. Biophys. Res.
Commun. 299, 268–272.
SUPPORTING INFORMATION AVAILABLE
Single turnover reactions under anaerobic conditions for H154N
and R179S; the anaerobic to aerobic transition for H154N; single
turnover reactions under aerobic conditions for H154N and
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R179S; binding of PQQ to mutant enzymes; PQQH reoxidation
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3. Itoh, S., Ohshiro, Y., and Agawa, T. (1986) Reaction of reduced PQQ
to PQQ for H154N and Y175S. This material is available free of
charge via the Internet at http://pubs.acs.org.
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(PQQH ) and molecular oxygen. Bull. Chem. Soc. Jpn. 59, 1911–1914.
4. Wulfsberg, G. (1991) Principles of Descriptive Inorganic Chemistry,
University Science Books, Sausalito, CA.
5. Klinman, J. P. (2007) How do enzymes activate oxygen without
inactivating themselves? Acc. Chem. Res. 40, 325–333.
6. Solomon, E. I., Chen, P., Metz, M., Lee, S. K., and Palmer, A. E.
(2001) Oxygen binding, activation, and reduction to water by copper
proteins. Angew. Chem., Int. Ed. 40, 4570–4590.
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