39NISR Research GRANT
2010 Research Grant

Studies on the novel menaquinone biosynthetic pathway andits application to anti-Helicobacter pylori drugs
Tohru DAIRI
Graduate School of Engineering, Hokkaido University

In prokaryotes, menaquinone (MK) is used for respiration. In Escherichia coli, MK is biosynthesized from chorismate by eight enzymes. However, we recently identified
an alternative pathway (the futalosine pathway), which operates in bacteria, including some pathogens, such as Helicobacter pylori and Campylobacter jejuni 1,2). However, thedetails of each of the biosynthetic steps in the futalosine
pathway remain unclear. In this study, we investigated thesecond step of the futalosine pathway, which is the conversion of futalosine (FL) into dehypoxanthinyl futalosine(DHFL) by MqnB (futalosine hydrolase, EC number,
EC3.2.2.26), in Acidothermus cellulolyticus, H. pylori andStreptomyces coelicolor. Although the mqn genes werescattered throughout the genomes in most of microorganisms, the mqn genes and other menaquinone-related genes
such as prenylation and methylation genes were clustered in
two loci on the genome of A. cellulolyticus, a thermophilic
actinobacteria. Interestingly, a gene for an adenosine deaminase ortholog (Acel_0264) exists in one of the clusters.
Considering that biosynthetically related genes are usuallyclustered in microorganism genomes, we can assume thatFig. 1.

aminodeoxyfutalosine (AFL) might be an intermediate in
the futalosine pathway operating in this strain. To examine
this possibility, we prepared the recombinant Acel_0264
gene product and incubated it with chemically synthesized
AFL. As expected, the protein converted AFL into FL. We
also examined whether the A. cellulolyticus MqnB protein,
the gene product of Acel_0106, could directly use AFL as asubstrate. However, we did not detect the formation ofDHFL, despite the fact that AFL and FL have very similar
structures. On the other hand, the recombinant MqnB
protein from Thermus thermophilus (the product ofTTHA0556) only reacted on FL. Judging from the genome
database of T. thermophilus, this strain may not have any
adenosine deaminases. Therefore, it was quite reasonable
that the T. thermophilus MqnB only accepted FL as a substrate.
We next searched for the MqnB ortholog in the genome
database of H. pylori strain 26695 using the A. cellulolyticus MqnB protein as a query. However, we did not find atypical MqnB ortholog with a significant estimated value(e-value), although the strain had an MqnC ortholog

An alternative menaquinone biosynthetic pathway (the futalosine pathway).

40(HP0656, e-value 4e81) and an MqnD ortholog (HP0152,e-value 3e16). The gene from strain 26695 encoding themost similar protein to the A. cellulolyticus MqnB proteinwas HP0089 (e-value, 0.18), which was annotated as a geneencoding a 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC: 3.2.2.9). Therefore, we prepared arecombinant protein encoded by HP0089 and incubated itwith FL; however, the protein did not act on FL. Therefore,
we assumed that AFL may be an intermediate in the futalosine pathway operating in H. pylori strain 26695. Therefore, the recombinant HP0089 gene product was incubatedwith AFL and the reaction product was analyzed by HPLC.
As expected, AFL was consumed and two products weredetected. The products were confirmed to be adenine andDHFL by LC-MS analysis. This result showed that AFL
was directly converted into DHFL. This direct conversion
without deamination was also in agreement with the observation that strain 26695 did not have an ortholog ofAcel_0264, which encodes an AFL deaminase.
In summary, three routes to the formation of DHFL in

the futalosine pathway were suggested to exist (Fig. 1)3). InT. thermophilus, FL might be directly formed from chorismate by MqnA and converted into DHFL. On the otherhand, AFL is likely formed from chorismate by MqnA in A.
cellulolyticus, S. coelicolor, and H. pylori. In the case of theformer two bacteria, AFL is thought to be converted to FL
by deaminases, which is subsequently metabolized into
DHFL. In contrast, AFL is most likely directly converted to
DHFL in H. pylori.
References
1)

Seto, H., Jinnai, Y., Hiratsuka, T., Fukawa, M., Furihata, K., Itoh, N.,
and Dairi, T. (2008) Studies on a new biosynthetic pathway formenaquinone. J. Am. Chem. Soc. 130: 5614–5615.
2) Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N.,
Seto, H., and Dairi, T. (2008) An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321: 1670–1673.
3) Arakawa, C., Kuratsu, M., Furihata, K., Hiratsuka, T., Itoh, N., Seto,H., and Dairi, T. (2011) Diversity of the early step of the futalosine
pathway. Antimicrob. Agents Chemother. 55: 913–916.