Significance of Phe-220 and Gln-221 in the Catalytic Mechanism of Farnesyl Diphosphate Synthase of Bucillus stearothermophilus

Koyama, Tanetoshi; Tajima, Masanori; Nishino, Tokuzo; Ogura, Kyozo

doi:10.1006/bbrc.1995.2022

Cited by 33 publications

(34 citation statements)

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“…The trans-type prenyltransferases all have two DDXXD motifs responsible for allylic substrate (FPP) and homoallylic substrate (IPP) binding (24). The Asp residues in the motif play essential role in Mg 2ϩ binding, and the substitution of these Asp residues with Ala led to the remarkable decrease of substrate affinity and turnover number (25,26). However, none of these motifs is found in the cis-type enzymes.…”

Section: Resultsmentioning

confidence: 99%

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl Pyrophosphate Synthase in Complex with Sulfate, Magnesium, and Triton

Chang¹,

Ko²,

Liang

et al. 2003

Journal of Biological Chemistry

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Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPPs from Escherichia coli is a dimer, and each subunit consists of 253 amino acid residues. The chain length of the product is modulated by a hydrophobic active site tunnel. In this paper, the crystal structure of E. coli UPPs was refined to 1.73 Å resolution, which showed bound sulfate and magnesium ions as well as Triton X-100 molecules. The amino acid residues 72-82, which encompass an essential catalytic loop not seen in the previous apoenzyme structure (Ko, T. Undecaprenyl pyrophosphate synthase (UPPs) 1 catalyzes the consecutive condensation reactions of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to form a lipid carrier to mediate bacterial peptidoglycan synthesis (1-3). This enzyme belongs to a family of prenyltransferases that make linear IPP condensation products with designate chain lengths (4). These enzymes are divided into trans-type and cis-type, which catalyze the trans-and cisdouble bond formation during each IPP condensation, respectively (5, 6). Unlike trans-prenyltransferases, which tend to make short and medium chain-length products ranging from C 15 to C 50 , UPPs and other cis-prenyltransferases mostly generate ՆC 55 long-chain products. Significant sequence homology has been found within the cis-and trans-prenyltransferases, but the two groups of enzymes do not share sequence similarity (7,8). Among the trans-prenyltransferases, the crystal structure of avian farnesyl pyrophosphate synthase has been solved almost a decade ago, and the mechanism has been elucidated (9). However, only recently have the first crystal structures of cis-prenyltransferase (UPPs) become available (10, 11), and they provide a template for modeling other cis-enzymes such as dehydrodolichyl pyrophosphate synthase from yeast and human and a polyprenyl pyrophosphate synthase discovered in Arabidopsis thaliana (12,13).UPPs from Escherichia coli is a dimer of identical subunits of 253 amino acids. The three-dimensional structure of the apoenzyme reveals an elongated tunnel-shaped active site crevice surrounded by two ␣-helices and four ␤-strands (11). Previous site-directed mutagenesis studies suggested that the substrates FPP and IPP are bound on top of the tunnel, and the farnesyl moiety of FPP migrates toward its bottom during product chain elongation (14 -16). The tunnel is sealed at the bottom by the side chain of . In the crystal structure of UPPs from Micrococcus luteus, a sulfate ion bound to a conserved structural P-loop represents the location of the pyrophosphate moiety of FPP (10).The previous E. coli UPPs crystal was grown using polyethylene glycol (PEG), and no sulfate ion was observed (11). Metal ion was not found in both apo-UPPs structures, although the enzyme requires Mg 2ϩ for activity. Two protein conformers of the E. coli UPPs were observed, i.e. one with a bou...

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Section: Resultsmentioning

confidence: 99%

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl Pyrophosphate Synthase in Complex with Sulfate, Magnesium, and Triton

Chang¹,

Ko²,

Liang

et al. 2003

Journal of Biological Chemistry

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“…It is suggested that Region HI, including the conserved Asn-Trp/Phe (NW/F) motif, plays an important role in the enzymatic catalysis and substrate binding for enzymatic cis-prenyl chain elongation. This motif is reminiscent of the Phe-Gln (FQ) motif of trans-prenyl chain elongating enzymes, which has been shown to be essential not only for the binding of allylic substrates but also for catalytic function (31). These motifs consisting of carbamoyl-and aromatic residues may be common and essential parts of the prenyl chain elongating machinery that triggers the consecutive condensation of IPP to produce polyprenyl chains stereospecifically.…”

Section: Random Pcr Mutagenesis Of the Upp Synthase Genementioning

confidence: 98%

Significance of Asn-77 and Trp-78 in the Catalytic Function of Undecaprenyl Diphosphate Synthase of Micrococcus luteus B-P 26

Fujikura

Zhang

Yoshizaki

et al. 2000

Journal of Biochemistry

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The primary structures of cis-prenyltransferases are completely different from those of trans-prenyltransferases. To obtain information about amino acid residues relating to catalytic function, random mutation of the undecaprenyl diphosphate synthase gene of Micrococcus luteus B-P 26 was carried out to construct a mutated gene library using an error-prone polymerase chain reaction. From the library, the mutants showing poor enzymatic activity were selected by the colony autoradiography method. Among 31 negative clones selected from 3,000 mutants, two clones were found to contain only one amino acid substitution at either Asn-77 or Trp-78. To determine the functional roles of these interesting residues, we prepared six mutated enzymes with substitutions at residues Asn-77 or Trp-78 by site-directed mutagenesis. Substitution of Asn-77 with Ala, Asp, or Gln resulted in a dramatic decrease in catalytic activity, but the K(m) values for both allylic and homoallylic substrates of these mutant enzymes were comparable to those of the wild-type. On the other hand, three Trp-78 mutants, W78I, W78R, and W78D, showed 5-20-fold increased K(m) values for farnesyl diphosphate but not for Z-geranylgeranyl diphosphate. However, these mutants showed moderate levels of enzymatic activity and comparable K(m) values for isopentenyl diphosphate to that of the wild-type. These results suggest that the Asn-Trp motif is involved in the binding of farnesyl diphosphate and enzymatic catalysis.

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“…The active site is located in the central cavity with two conserved Asp-rich motifs facing each other on opposite walls. These motifs have been demonstrated to be involved in substrate binding and catalysis via coordination with Mg 2+ , hence the requirement of this avian FPP synthase and other IDSs for this cofactor (Marrero et al, 1992;Joly and Edwards, 1993;Song and Poulter, 1994;Koyama et al, 1994Koyama et al, , 1995Koyama et al, , 1996. In PaIDS1, the first Asp-rich region spans amino acid residues 170 to 183 and is located in helix D and an adjacent loop, while the second region spans residues 309 to 313 and is located in helix H. These two helices, as well as helix F1, form a significant portion of the active site (depicted in Fig.…”

Section: +mentioning

confidence: 99%

“…2). Site-directed mutagenesis studies found that most of the Asp residues in these two highly conserved motifs are critical for substrate binding and catalysis (Koyama et al, 1995(Koyama et al, , 1996. The amino acid sequence features that determine the chain length of the product have been studied by random chemical mutagenesis and experiments on an avian FPP synthase, from which an x-ray structure has been obtained (Tarshis et al, 1994).…”

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confidence: 99%

A Bifunctional Geranyl and Geranylgeranyl Diphosphate Synthase Is Involved in Terpene Oleoresin Formation inPicea abies

et al. 2009

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The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C10) and diterpenes (C20). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C10), farnesyl diphosphate (C15), and geranylgeranyl diphosphate (C20) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C10) and geranylgeranyl diphosphate (C20). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

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Significance of Phe-220 and Gln-221 in the Catalytic Mechanism of Farnesyl Diphosphate Synthase of Bucillus stearothermophilus

Cited by 33 publications

References 0 publications

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl Pyrophosphate Synthase in Complex with Sulfate, Magnesium, and Triton

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl Pyrophosphate Synthase in Complex with Sulfate, Magnesium, and Triton

Significance of Asn-77 and Trp-78 in the Catalytic Function of Undecaprenyl Diphosphate Synthase of Micrococcus luteus B-P 26

A Bifunctional Geranyl and Geranylgeranyl Diphosphate Synthase Is Involved in Terpene Oleoresin Formation inPicea abies

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