Refolding and Characterization of a Yeast Dehydrodolichyl Diphosphate Synthase Overexpressed in Escherichia coli

Chang, Shuenn‐Yih; Tsai, Pei Chun; Tseng, Chien Sheng; Liang, Po‐Huang

doi:10.1006/prep.2001.1511

Cited by 14 publications

(9 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Analogously to the two DDxxD motifs in the trans ‐prenyltransferases, site‐directed mutagenesis studies of UPPS indicate that Asp and Glu play a significant role in IPP binding and activity probably through co‐ordination with Mg 2+ . The IPP condensation kinetics for UPPS, DDPPS ( cis ‐type) and OPPS ( trans ‐type) had been measured for comparison of IPP condensation catalyzed by cis ‐IPPS and trans ‐IPPS [76–78]. These experiments were performed under the enzyme single‐turnover condition using a rapid‐quench instrument, because the slow release of the large hydrophobic product limits IPPS catalysis under steady‐state conditions.…”

Section: Class I: Isoprenyl Pyrophosphate Synthases (Ippss)mentioning

confidence: 99%

Structure, mechanism and function of prenyltransferases

Liang

Wang

2002

European Journal of Biochemistry

Self Cite

391

367

View full text Add to dashboard Cite

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.

show abstract

Section: Class I: Isoprenyl Pyrophosphate Synthases (Ippss)mentioning

confidence: 99%

Structure, mechanism and function of prenyltransferases

Liang

Wang

2002

European Journal of Biochemistry

Self Cite

391

367

View full text Add to dashboard Cite

show abstract

“…UPPs along with other cis ‐prenyltransferases catalyze cis ‐double bond formation during IPP condensation but synthesize different chain‐length products (Ogura and Koyama 1998). Dehydrodolichyl pyrophos‐phate synthases for glycoprotein lipid carrier biosynthesis generate products with chain lengths ranging from C 55 to C 120 (Sato et al 1999; Chang et al 2001). Rubber prenyl‐transferase synthesizes a huge polymer containing thousands of IPP units (Cornish 2001).…”

mentioning

confidence: 99%

Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies

Chang

Chen

et al. 2004

Protein Science

View full text Add to dashboard Cite

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to form a 55-carbon long-chain product. We previously reported the crystal structure of the apo-enzyme from Escherichia coli and the structure of UPPs in complex with sulfate ions (resembling pyrophosphate of substrate), Mg 2+ , and two Triton molecules (product-like). In the present study, FPP substrate was soaked into the UPPs crystals, and the complex structure was solved. Based on the crystal structure, the pyrophosphate head group of FPP is bound to the backbone NHs of Gly29 and Arg30 as well as the side chains of Asn28, Arg30, and Arg39 through hydrogen bonds. His43 is close to the C2 carbon of FPP and may stabilize the farnesyl cation intermediate during catalysis. The hydrocarbon moiety of FPP is bound with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the ␣3 helix. The binding mode of FPP in cis-type UPPs is apparently different from that of trans-type and many other prenyltransferases which utilize Asp-rich motifs for substrate binding via Mg 2+ . The new structure provides a plausible mechanism for the catalysis of UPPs.Keywords: prenyltransferase; farnesyl pyrophosphate; isopentenyl pyrophosphate; crystal structure; substrate binding; metal ion (Sato et al. 1999;Chang et al. 2001). Rubber prenyltransferase synthesizes a huge polymer containing thousands of IPP units (Cornish 2001). In contrast, trans-prenyltransferases, which generate trans-double bonds in IPP condensation, synthesize shorter chain-length products, C 15 -C 50 (Chen et al. 1994; Wang and Ohuma 2000). Abbreviations: UPPs, undecaprenyl pyrophosphate synthase; IPP, isopentenyl pyrophosphate; FPP, farnesyl pyrophosphate; UPP, undecaprenyl pyrophosphate; FPPs, farnesyl pyrophosphate synthase; GPP, geranyl pyrophosphate; TLC, thin-layer chromatography; NiNTA, nickel nitrilo-triacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; FTase, farnesyltransferase.Article and publication are at http://www.proteinscience.org/cgi

show abstract

“…In papers dealing with refolding proteins other than antibodies or their fragments, there are several methods, a 'functional assay', in addition to the absorbance (A 280 ) as usual, as described above [19], such as the enzymatic activity of an authentic sample [20], recovery yield of the cell lysate [21], or the receptor-ligand binding assay [22].…”

Section: Discussionmentioning

confidence: 99%