Type II Isopentenyl Diphosphate Isomerase from Synechocystis sp. Strain PCC 6803

ABSTRACT Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His6-tagged protein was produced in Escherichia coli and purified by Ni2+ chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg2+ for activity; K m IPP was 52 μM, and k cat IPP was 0.23 s−1.

Download Full-text

Escherichia coli Open Reading Frame 696 Is idi, a Nonessential Gene Encoding Isopentenyl Diphosphate Isomerase

Journal of Bacteriology ◽

10.1128/jb.181.15.4499-4504.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4499-4504 ◽

Cited By ~ 108

Author(s):

Frederick M. Hahn ◽

Anthony P. Hurlburt ◽

C. Dale Poulter

Keyword(s):

Escherichia Coli ◽

Mevalonate Pathway ◽

Open Reading Frame ◽

Restrictive Temperature ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

Reading Frame ◽

E Coli ◽

Isoprene Unit ◽

Nonessential Gene

ABSTRACT Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3′-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44°C and FH12 lacking the plasmid grew on minimal medium, thereby establishing thatidi is a nonessential gene. Although theV max of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance inKm s. The E. coli protein requires Mg2+ or Mn2+ for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.

Download Full-text

Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

Journal of Bacteriology ◽

10.1128/jb.178.3.619-624.1996 ◽

1996 ◽

Vol 178 (3) ◽

pp. 619-624 ◽

Cited By ~ 28

Author(s):

F M Hahn ◽

J A Baker ◽

C D Poulter

Keyword(s):

Gene Cluster ◽

Rhodobacter Capsulatus ◽

Open Reading Frame ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

Reading Frame ◽

Photosynthesis Gene

Download Full-text

The Synechocystis sp. PCC 6803 open reading frame slr0201 that is homologous to sdhC from Archaea codes for a [2Fe-2S] protein

10.1101/2021.09.23.461530 ◽

2021 ◽

Author(s):

Fusheng Xiong ◽

Russell LoBrutto ◽

Wim F. J. Vermaas

Keyword(s):

Sequence Similarity ◽

Immunoblot Analysis ◽

Open Reading Frame ◽

Heterodisulfide Reductase ◽

Redox Titration ◽

High Sequence Similarity ◽

Reading Frame ◽

Midpoint Potential ◽

Synechocystis Sp ◽

Pcc 6803

A hypothetical protein encoded by Synechocystis sp. PCC 6803 open reading frame slr0201 shows high sequence similarity to the C subunit of a group of unusual succinate dehydrogenases found in some archaeal species. Slr0201 was originally annotated as HdrB, the B subunit of heterodisulfide reductase, but appears to be SdhC instead. This protein was overexpressed in E. coli by cloning the PCR-derived slr0201 open reading frame into a pET16b-based expression vector. The overproduced Slr0201 accumulated predominantly in inclusion bodies with an apparent molecular mass of 33 kDa. The protein contained at least one [2Fe-2S] cluster based on UV-visible absorbance and CD spectra and EPR spectroscopy, in conjunction with stoichiometric analysis of protein-bound iron and sulfur content. Redox titration showed a midpoint potential (Em) of + 17 mV at pH 7.0, which is consistent with Slr0201 serving a role in transferring electrons between succinate and plastoquinone. Slr0201 was also overproduced in Synechocystis sp. PCC 6803 by introducing an additional, His-tagged slr0201 into the Synechocystis genome replacing psbA3, creating the slr0201+-His overexpression strain. Immunoblot analysis shows that Slr0201 is membrane-associated in the wild type. However, in the Slr0201+-His strain, immunoreaction occurred in both the membrane and soluble fractions, possibly as a consequence of processing near the N-terminus. The results obtained with Slr0201 are discussed in the light of one of the cyanobacterial SdhB subunits, which shares redox commonalities with archaeal SdhB.

Download Full-text

Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin

Journal of Bacteriology ◽

10.1128/jb.183.20.6085-6094.2001 ◽

2001 ◽

Vol 183 (20) ◽

pp. 6085-6094 ◽

Cited By ~ 63

Author(s):

Tohru Dairi ◽

Yoshimitsu Hamano ◽

Tomohisa Kuzuyama ◽

Nobuya Itoh ◽

Kazuo Furihata ◽

...

Keyword(s):

Escherichia Coli ◽

Sequence Analysis ◽

Mevalonate Pathway ◽

Open Reading Frame ◽

Upstream Region ◽

Constitutive Promoter ◽

Isopentenyl Diphosphate ◽

Geranylgeranyl Diphosphate ◽

Reading Frame ◽

Open Reading Frame 2

ABSTRACT 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 theermE* 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.

Download Full-text