SEQUENCE-ANALYSIS OF PQQ GENES REQUIRED FOR BIOSYNTHESIS OF PYRROLOQUINOLINE QUINONE IN METHYLOBACTERIUM-EXTORQUENS AM1 AND THE PURIFICATION OF A BIOSYNTHETIC INTERMEDIATE
H. Toyama et al., SEQUENCE-ANALYSIS OF PQQ GENES REQUIRED FOR BIOSYNTHESIS OF PYRROLOQUINOLINE QUINONE IN METHYLOBACTERIUM-EXTORQUENS AM1 AND THE PURIFICATION OF A BIOSYNTHETIC INTERMEDIATE, Microbiology, 143, 1997, pp. 595-602
Methylobacterium extorquens AM1 produces pyrroloquinoline quinone (PQQ
), the prosthetic group of methanol dehydrogenase. Two gene clusters h
ave been shown to be required for PQQ biosynthesis in this micro-organ
ism and complementation analysis has identified seven pqq genes, pqqDG
CBA and pqqEF. The DNA sequence of pqqDGC' was reported previously. Th
is paper reports the sequence of the genomic region corresponding to p
qqC'BA. For consistency, the nomenclature of pqq genes in Klebsiella p
neumoniae will be followed. The new nomenclature for pqq genes of M. e
xtorquens AM1 is pqqABCDE and pqqFG. In the genomic region sequenced i
n this study, two open reading frames were found. One of these encodes
PqqE, which showed high identity to analogous pqq genes in other bact
eria. PqqE also showed identity to MoaA and NifB in the N-terminal reg
ion, where a conserved CxxxCxYC sequence was identified. The sequence
of the second open reading frame covered both the pqqC and pqqD region
s, suggesting that both functions were encoded by this gene. It is pro
posed to designate this gene pqqC/D. The deduced amino acid sequence o
f the pqqC/D 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 regio
n. A fragment of M. extorquens AM1 DNA containing only pqqC/D produced
a protein of 42 kDa in Escherichia coli, which corresponds to the siz
e of the deduced amino acid sequence of PqqC/D, confirming the absence
of a separate pqqD. This genomic region complemented the growth of pq
qC 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 b
iosynthesis, which was converted to PQQ by incubation with a crude ext
ract from E. coil cells expressing PqqC/D. The intermediate was found
in both crude extract and culture supernatant, and it was purified fro
m the crude extract. The PqqC/D enzyme reaction appeared to require mo
lecular oxygen and reduced nicotinamide adenine dinucleotides.