Comparison of the enzymatic properties of ent-copalyl diphosphate synthases in the biosynthesis of phytoalexins and gibberellins in rice

The rice genome contains two ent-copalyl diphosphate synthase genes: OsCPS1 acts in gibberellin (phytohormone) biosynthesis, and OsCPS2/OsCyc2 acts in the synthesis of oryzalexins A-F and phytocassanes A-E (phytoalexins). We characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 fused with a tag-protein at the N-terminus, and compared them to those of OsCPS1. Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH, optimal temperature, divalent cation requirement, and kinetic values for the geranylgeranyl diphosphate (GGDP) substrate, were almost the same as those of OsCPS1. However, OsCPS2/OsCyc2 activity was not inhibited by 50-60 muM GGDP substrate, by which the OsCPS1 activity was inhibited. Furthermore, the OsCPS1 activity exhibited approximately 70% inhibition by 100 muM Amo-1618 (a gibberellin biosynthetic inhibitor), whereas the OsCPS2/OsCyc2 activity exhibited approximately 10% inhibition. These results indicate that the properties of OsCPS2/OsCyc2 were partially different from those of OsCPS1, although OsCPS2/OsCyc2 catalyzes the same reaction step as OsCPS1.     

Engineering of Phenylacetaldehyde Reductase for Efficient Substrate Conversion in Concentrated 2-Propanol

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.     

Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.

Summary We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employs the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pM0116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.     

Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

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.     

Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

Aims:  The aim of this study was to isolate a thermotolerant micro‐organism that produces polyhydroxyalkanoates (PHAs) composed of medium‐chain‐length (mcl) HA units from a biodiesel fuel (BDF) by‐product as a carbon source. Methods and Results:  We successfully isolated a thermotolerant micro‐organism, strain SG4502, capable to accumulate mcl‐PHA from a BDF by‐product as a carbon source at a cultivation temperature of 45°C. The strain could also produce mcl‐PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55°C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions:  A novel thermotolerant bacterium capable to accumulate mcl‐PHA from a BDF by‐product was successfully isolated. Significance and Impact of the Study:  A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical‐based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by‐product and produces PHA at high temperature, would be very useful for practical application in industry.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20–29% identity with zinc-containing, NAD⁺-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD⁺- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity. Received: 5 March 1999 / Received last revision: 10 May 1999 / Accepted: 16 May 1999     

The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD⁺ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD⁺ turnover, which makes NAD⁺ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD⁺ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD⁺ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.The cyanoguanidinopyridine GMX1778 (previously known as CHS828) is the active form of the prodrug GMX1777 and has potent antitumor activity in vitro and in vivo against cell lines derived from several different tumor origins (11). The antitumor activity of GMX1778 has been widely studied since its discovery (1, 11, 19-21, 24), but positive identification of the molecular target and the mechanism of action of GMX1778 has been elusive. Here, we demonstrate that GMX1778 exerts its antitumor activity via its potent and selective antagonism of NAD⁺ biosynthesis. GMX1777 is currently being assessed in phase I clinical trials for treatment of patients with refractory solid tumors.The pyridine nucleotide NAD⁺ plays a major role in the regulation of several essential cellular processes (7, 22, 25, 38). In addition to being a biochemical cofactor for enzymatic redox reactions involved in cellular metabolism, including ATP production, NAD⁺ is important in diverse cellular pathways responsible for calcium homeostasis (17), gene regulation (5), longevity (18), genomic integrity (33), and apoptosis (36). Cancer cells exhibit a significant dependence on NAD⁺ for support of the high levels of ATP production necessary for rapid cell proliferation. They also consume large amounts of this cofactor via reactions that utilize poly(ADP) ribosylation, including DNA repair pathways (10, 37, 39).In eukaryotes, the biosynthesis of NAD⁺ occurs via two biochemical pathways: the de novo pathway, in which NAD⁺ synthesis occurs through the metabolism of l-tryptophan via the kynurenine pathway, and the salvage pathway. The NAD⁺ salvage pathway can use either nicotinamide (niacinamide) (NM) or nicotinic acid (niacin) (NA) (via the Preiss-Handler pathway) as a substrate for NAD⁺ production. Saccharomyces cerevisiae species predominantly use NA as the substrate for NAD⁺ biosynthesis, through the deamidation of NM by the nicotinamidase PNC1 (25). However, mammalian cells do not express a nicotinamidase enzyme and use NM as the preferred substrate for the NAD⁺ salvage pathway. The mammalian NAD⁺ biosynthesis salvage pathway using NM is composed of NA phosphoribosyltransferase (NAMPT), which is the rate-limiting and penultimate enzyme that catalyzes the phosphoribosylation of NM to produce nicotinamide mononucleotide (NMN) (27, 29). NMN is subsequently converted to NAD⁺ by NMN adenyltransferases (NMNAT). The gene encoding NAMPT was originally identified as encoding a cytokine named pre-B-cell colony-enhancing factor (PBEF1) (30). NAMPT was also identified as a proposed circulating adipokine named visfatin (thought to be secreted by fat cells) and was suggested to function as an insulin mimetic; however, this role of NAMPT currently remains controversial (8). In mice, NAMPT has been shown to act as a systemic NAD⁺ biosynthetic enzyme that regulates insulin secretion from β cells (28). The molecular structure of NAMPT from human (15), rat (16) and mouse (35) tissue, containing either NMN or the inhibitor APO866, have been determined by X-ray crystallography. These structures revealed that NAMPT is a dimeric type II phosphoribosyltransferase.Here, we report that the anticancer compound GMX1778 is a specific inhibitor of NAMPT in vivo and in vitro and is itself a substrate for the enzyme. Phosphoribosylated GMX1778 inhibits NAMPT as potently as GMX1778 but is preferentially retained within cells. Finally, we have identified a novel anticancer strategy utilizing NA rescue of GMX1778 cytotoxicity to increase the therapeutic index of GMX1777 activity in tumors that are deficient in NA phosphoribosyltransferase 1 (NAPRT1).