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Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC.

ABSTRACT: Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the presence of PQQ. These putative PQQ biosynthesis mutants have been complemented by using previously isolated clones containing M. extorquens AM1 DNA, which were known to contain genes necessary for oxidation of methanol to formaldehyde (mox genes). Subcloning and transposon mutagenesis experiments have assigned these mutants to five complementation groups in two gene clusters. Representatives of each complementation group were shown to lack detectable PQQ in the growth medium and in cell extracts and to contain methanol dehydrogenase polypeptides that were inactive. Therefore, these mutants all appear to be defective in PQQ biosynthesis. PQQ biosynthesis mutants of Methylobacterium organophilum DSM 760 and M. organophilum XX were complemented by using M. extorquens AM1 subclones, and PQQ biosynthesis mutants of M. extorquens AM1 and M. organophilum XX were complemented by using M. organophilum DSM 760 subclones. This analysis suggested that a total of six PQQ biosynthesis complementation groups were present in M. extorquens AM1 and M. organophilum DSM 760. A 2-kb M. extorquens AM1 DNA fragment that complemented the MoxO class of PQQ biosynthesis mutants was sequenced and found to contain two complete open reading frames and the N-terminal sequence of a third. These genes designated pqqDGC, had predicted gene products with substantial similarity to the gene products of corresponding pqq genes in Acinetobacter calcoaceticus and Klebsiella pneumoniae. pqqD encodes a 29-amino-acid peptide which contains a tyrosine residue and glutamate residue that are conserved in the equivalent peptides of K. pneumoniae, PqqA (23 amino acids), and A. calcoaceticus, PqqIV (24 amino acids), and are thought to be the precursors for PQQ biosynthesis. The organizations of a cluster of five PQQ biosynthetic genes appear to be similiar in four different bacteria (M. extorquens AM1, M. organophilum DSM 760, K. pneumoniae, and A. calcoaceticus). Our results show that a total of seven pqq genes are present in M. extorquens AM1, and these have been designated pqqDGCBA and pqqEF.

SUBMITTER: Morris CJ

PROVIDER: S-EPMC205263 | biostudies-other | 1994 Mar

REPOSITORIES: biostudies-other

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Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC.

Morris C J CJ Biville F F Turlin E E Lee E E Ellermann K K Fan W H WH Ramamoorthi R R Springer A L AL Lidstrom M E ME

Journal of bacteriology 19940301 6

Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the prese ...[more]

PMID: 8132470

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Project description:Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La3+). ExaF was purified from cells grown with both calcium (Ca2+) and La3+ and with Ca2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La3+ over Ca2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca2+-plus-La3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca2+-only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La3+ is present, expanding the role of lanthanides to multicarbon metabolism.ImportanceExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Dataset Information

Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC.

Publications

Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC.

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