Sequence analysis of pqq genes required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1 and the purification of a biosynthetic intermediate

Abstract

Methylobacterium extorquens AM1 produces pyrroloquinoline quinone (PQQ), the prosthetic group of methanol dehydrogenase. Two gene clusters have been shown to be required for PQQ biosynthesis in this micro-organism and complementation analysis has identified seven pqq genes, pqqDGCBA and pqqEF. The DNA sequence of pqqDGC' was reported previously. This paper reports the sequence of the genomic region corresponding to pqqC'BA. For consistency, the nomenclature of pqq genes in Klebsiella pneumoniae will be followed. The new nomenclature for pqq genes of M. extorquens AM1 is pqqABCDE and pqqFG. In the genomic region sequenced in this study, two open reading frames were found. One of these encodes PqqE, which showed high identity to analogous pqq genes in other bacteria. PqqE also showed identity to MoaA and NifB in the N-terminal region, where a conserved CxxxCxYC sequence was identified. The sequence of the second open reading frame covered both the pqqC and pqqD regions, suggesting that both functions were encoded by this gene. It is proposed to designate this gene pqqCID. The deduced amino acid sequence of the pqqCID product showed identity to PqqC of K. pneumoniae and Pqql of Acinetobacter calcoaceticus in the N-terminal region, and to PqqD of K. pneumoniae and Pqqll of A. calcoaceticus in the C-terminal region. A fragment of M. extorquens AM1 DNA containing only p9qCID produced a protein of 42 kDa in Escherichia coli, which corresponds to the size of the deduced amino acid sequence of PqqUD, confirming the absence of a separate pqqD. This genomic region complemented the growth of pqqC mutants of M. extorquens AM1 and Methylobacterium organophilum DSM 760 on methanol. As previously reported for pqq genes of K. pneumoniae, a pqqC mutant of M. extorquens AM1 produced an intermediate of PQQ biosynthesis, which was converted to PQQ by incubation with a crude extract from E. coli cells expressing PqqUD. The intermediate was found in both crude extract and culture supernatant, and it was purified from the crude extract. The PqqUD enzyme reaction appeared to require molecular oxygen and reduced nicotinamide adenine dinucleotides.

PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway

“…PqqD has been shown, through gene knock-out and bioinformatic studies, to be an essential component for PQQ biosynthesis (8,14,15). The crystal structure for XcPqqD indicates that PqqD is a dimer and may form a scaffold for a larger macromolecular complex (15).…”

“…Certain ribosomally derived products rely on radical S-adenosylmethionine enzymes (herein referred to as RS proteins) to catalyze the formation of ␣-carbon thioether bonds or carbon-carbon bonds (8,9). In the subtilosin and the pyrroloquinoline quinone (PQQ) 2 pathways, the RS protein also contains a C-terminal extension referred to as a SPASM domain (subtilosin, PQQ, anaerobic sulfatase maturating enzyme, and mycofactocin).…”

Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone

“…PqqC from K. pneumoniae (NCBI accession no. X58778) was expressed in E. coli and purified as described elsewhere (19).Single-Turnover Kinetics, Peroxide Formation, and Oxygen Uptake.The substrate for PqqC was purified from a pqqC knock-out mutant of M. extorquens AM1 as described elsewhere (17,18). All experiments were performed in 0.1 M potassium phosphate buffer (pH 8.0) at 20°C, and all data fitting was done by using KALEIDAGRAPH (Synergy Software, Reading, PA).…”

“…It has been shown that the PqqC reaction is accelerated in the presence of molecular oxygen (17) and that it requires NADPH and an uncharacterized activating factor (16) for sustained catalytic activity. Most recently, we elucidated the structure of the PqqC substrate, allowing the overall reaction catalyzed by PqqC to be inferred (18).…”

Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

“…The gene mxaD encodes the 17 kDa periplasmic protein that directly or indirectly stimulates the interaction between methanol dehydrogenase and cytochrome c L (Toyama et al, 2003), and the other genes in this region are involved in either assembly or regulatory functions (Anthony, 2000;Lidstrom & Tsygankov, 1991). Two other regulatory genes (mxbDM) (Springer et al, 1997) and genes required for synthesis of the PQQ prosthetic group, pqqABC/DE (Morris et al, 1994;Toyama et al, 1997), are found at the mxb locus. The other two known pqq genes (pqqFG) reside within another cluster in the genome (Springer et al, 1996).…”