Haruo Seto

Fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis

Fosmidomycin inhibited 1-deoxy-d-xylulose 5-phosphate reductoisomerase pathway for terpenoid biosynthesis with IC50 of 8.2 nM.

Diversity of the biosynthesis of the isoprene units.

This review covers the biosynthesis of the starter units of terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the nonmevalonate pathway together with a new enzyme involved in the conversion of IPP and DMAPP, i.e type 2 IPP isomerase. The biosynthesis of terpenoids produced by actinomycetes is also reviewed. 117 references are cited.

Direct formation of 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate by 1-deoxy-d-xylulose 5-phosphate reductoisomerase, a new enzyme in the non-mevalonate pathway to isopentenyl diphosphate

Abstract 1-Deoxy- d -xylulose 5-phosphate is biotransformed to 2-C-methyl- d -erythritol 4-phosphate in a single step in the presence of NADPH by a new recombinant enzyme named 1-deoxy- d -xylulose 5-phosphate reductoisomerase purified from Escherichia coli .

An Alternative Menaquinone Biosynthetic Pathway Operating in Microorganisms

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.

Formation of 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol from 2-C-methyl-d-erythritol 4-phosphate by 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase, a new enzyme in the nonmevalonate pathway

2-C-Methyl-d-erythritol 4-phosphate is transformed to 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol in the presence of cytidine 5′-triphosphate by a novel Escherichia coli enzyme, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase, involved in the nonmevalonate pathway.

Studies on the nonmevalonate pathway: conversion of 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol to its 2-phospho derivative by 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase

Abstract A nonmevalonate pathway intermediate, 4-(cytidine 5′-diphospho)-2- C -methyl- d -erythritol, is transformed to its 2-phospho-derivative in the presence of ATP by a novel Escherichia coli enzyme, 4-(cytidine 5′-diphospho)-2- C -methyl- d -erythritol kinase.

Studies on the nonmevalonate pathway: formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate from 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol

Abstract 2-Phospho-4-(cytidine 5′-diphospho)-2- C -methyl- d -erythritol was transformed to 2- C -methyl- d -erythritol 2,4-cyclodiphosphate by a novel Escherichia coli enzyme involved in the nonmevalonate pathway.

Functional Analysis of Eubacterial Diterpene Cyclases Responsible for Biosynthesis of a Diterpene Antibiotic, Terpentecin

Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. Bacteriol. 183, 6085–6094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.

Structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in a quaternary complex with a magnesium ion, NADPH and the antimalarial drug fosmidomycin

The crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg(2+), NADPH and fosmidomycin was solved at 2.2 A resolution. DXR is the key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg(2+) was shifted by NADPH in the active site.

Mechanistic Studies of HPP Epoxidase: Configuration of the Substrate Governs Its Enzymatic Fate

Non-heme iron-dependent enzymes are an important class of catalysts involved in many biological transformations of medical, pharmaceutical, and environmental significance. Although considerable progress has been made in our understanding of their catalyses, characterization of the metal centers and the modes of dioxygen activation remain a challenge because of the great structural and mechanistic diversity found among these enzymes.[1] Recently, HPP epoxidase,[2] an enzyme in the biosynthetic pathway of the antibiotic fosfomycin, was recognized as a new member of this enzyme family.[3] This epoxidase performs the final transformation in the fosfomycin biosynthesis by converting (S)-2hydroxypropylphosphonic acid (HPP, (S)-1) to (1R,2S)-epoxypropylphosphonic acid (2), also known as fosfomycin COMMUNICATIONS