In microorganisms, menaquinone is an obligatory component of the electron-transfer pathway. It is derived from chorismate by seven enzymes in Escherichia coli. However, a bioinformatic analysis of whole genome sequences has suggested that some microorganisms, including pathogenic species such as Helicobacter pylori and Campylobacter jejuni, do not have orthologs of the men genes, even though they synthesize menaquinone. We deduced the outline of this alternative pathway in a nonpathogenic strain of Streptomyces by bioinformatic screening, gene knockouts, shotgun cloning with isolated mutants, and in vitro studies with recombinant enzymes. As humans and commensal intestinal bacteria, including lactobacilli, lack this pathway, it represents an attractive target for the development of chemotherapeutics.

We recently demonstrated that the futalosine pathway was operating in some bacteria for the biosynthesis of menaquinone and that futalosine was converted into dehypoxanthinyl futalosine (DHFL) by an MqnB of Thermus thermophilus. In this study, we found that aminodeoxyfutalosine, which has adenine instead of hypoxanthine in futalosine, was directly converted into DHFL by an MqnB of Helicobacter pylori. Therefore, this step is potentially an attractive target for the development of specific anti-H. pylori drugs.In prokaryotes, menaquinone (MK) is used for respiration. In Escherichia coli, MK is biosynthesized from chorismate by eight enzymes (4). However, we recently identified an alternative pathway (the futalosine pathway shown in Fig. 1) (2, 3, 5), which operates in bacteria, including some pathogens, such as Helicobacter pylori, Campylobacter jejuni, Chlamydia trachomatis, and Leptospira borgpetersenii. Disruption of this pathway in Streptomyces coelicolor has been shown to lead to the inhibition of bacteriostatic growth. We also showed that the Mqn genedisrupted S. coelicolor mutants required more than 100 g/ml menaquinone for their growth. Because we do not consume a diet containing this much menaquinone, the essential amount of MK is not supplied from our diet if the futalosine pathway is blocked. Moreover, humans and commensal intestinal bacteria, including lactobacilli, lack the futalosine pathway. Taking these results together, we think that the futalosine pathway is a potential attractive target for the development of specific anti-H. pylori drugs. However, the details of each of the biosynthetic steps in the futalosine pathway remain unclear. In this study, we investigated the second step of the futalosine pathway, which is the conversion of futalosine (FL) into dehypoxanthinyl futalosine (DHFL) by MqnB (futalosine hydrolase [EC 3.2.2.26]) (3), in Acidothermus cellulolyticus, H. pylori, and S. coelicolor. We found that aminodeoxyfutalosine (AFL), which has adenine instead of hypoxanthine in FL, was an intermediate in these microorganisms.To ascertain the details of each step in the futalosine pathway, we selected the second step catalyzed by MqnB, since FL, the substrate of MqnB, was the only compound that we could readily prepare from the culture broth of the MqnB-disrupted mutant (2). We first investigated the distribution of the futalosine pathway and the organization of the Mqn genes in microorganisms whose genomes had been completely sequenced.In almost all microorganisms, the Mqn genes were scattered throughout the genome. However, in A. cellulolyticus, a thermophilic actinobacterium, the Mqn genes, and other menaquinone-related genes, such as prenylation and methylation genes, were clustered in two loci (1). One cluster contained Acel_0105 and Acel_0106 genes, which encoded orthologs of MqnC and MqnB, respectively. The other was composed of 11 genes (Acel_0255 to Acel_0266) encoding MqnA (Acel_0261) and MqnC (Acel_0263) orthologs. To determine whether Acel_0106 did indeed encode MqnB, we prepare...

The biosynthetic gene cluster of antifungal agent jawsamycin (FR-900848) has been identified by heterologous expression. A series of gene inactivations and in vitro and in vivo analysis of key enzymes in the biosynthetic pathway established their functions. A novel mechanism involving a radical S-adenosyl methionine (SAM) cyclopropanase collaborating with an iterative polyketide synthase is proposed for the construction of the unique polycyclopropanated backbone. Our reconstitution system sets the stage for studying the catalytic mechanism of this intriguing contiguous cyclopropanation.

Menaquinone is biosynthesized from chorismate via a new pathway involving 1,4-dihydroxy-6-naphthoate in Streptomyces and presumably in pathogenic bacteria Helicobacter and Campylobacter.

Quinocarcin and SF-1739, potent antitumor antibiotics, share a common tetracyclic tetrahydroisoquinoline (THIQ)-pyrrolidine core scaffold. Herein, we describe the identification of their biosynthetic gene clusters and biochemical analysis of Qcn18/Cya18 generating the previously unidentified extender unit dehydroarginine, which is a component of the pyrrolidine ring. ATP-inorganic pyrophosphate exchange experiments with five nonribosomal peptide synthetases (NRPSs) enabled us to identify their substrates. On the basis of these data, we propose that a biosynthetic pathway comprising a three-component NRPS/MbtH family protein complex, Qcn16/17/19, plays a key role in the construction of tetracyclic THIQ-pyrrolidine core scaffold involving sequential Pictet-Spengler and intramolecular Mannich reactions. Furthermore, data derived from gene inactivation experiments led us to propose late-modification steps of quinocarcin.

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Jawsamycin is a polyketide-nucleoside hybrid with a unique polycyclopropane moiety on a single polyketide chain. The unexpected isolation of cyclopropane deficient jawsamycin analogs allowed us to propose a stepwise cyclopropanation mechanism for the enzymatic synthesis of this polyketide. The concise timing of the cyclopropanation could be regulated by a delicate balance between reaction rates of the condensation and cyclopropanation reactions.

Ferrichromes are a family of fungal siderophores with cyclic hexapeptide structures. Most fungi produce one or two ferrichrome-type siderophores. Acremonium persicinum MF-347833 produces ferrichrome-like potent Trojan horse antifungal antibiotics ASP2397 and AS2488053, the aluminum-and iron-chelating forms of AS2488059, respectively. Here, we show by gene sequencing followed by gene deletion experiments that A. persicinum MF-347833 possesses two nonribosomal peptide synthetase genes responsible for AS2488059 and ferricrocin assembly. AS2488059 was produced under iron starvation conditions and excreted into the media to serve as a defense metabolite and probably an iron courier. In contrast, ferricrocin was produced under iron-replete conditions and retained inside the cells, likely serving as an iron-sequestering molecule. Notably, the phylogenetic analyses suggest the different evolutionary origin of AS2488059 from that of conventional ferrichrome-type siderophores. Harnessing two ferrichrome-type siderophores with distinct biological properties may give A. persicinum a competitive advantage for surviving the natural environment.