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PubMed2003

Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis.

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.

Alkyl and Aryl Transferases↗
PubMed1999

Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution.

In the synthesis of isoprenoids, isoprenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce a variety of prenyl diphosphates with well-defined chain lengths. Site-directed mutagenesis in conjunction with X-ray crystallographic studies have identified specific amino acid residues responsible for chain-length determination. Simple combinations of these residues within a characteristic motif are not only sufficient to confer product specificities to all isoprenyl diphosphate synthases but represent structural features that reflect the enzyme family's evolutionary course.

Alkyl and Aryl Transferases↗
PubMed2002

Coupling of isoprenoid triflates with organoboron nucleophiles: synthesis and biological evaluation of geranylgeranyl diphosphate analogues.

The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methylboronic acid into a vinyl triflate. This has led to a concise synthesis of all-trans-geranylgeraniol, with the key step being the palladium-catalyzed, silver-mediated methylation of triflate to give ethyl geranylgeranoate. This coupling protocol has also been used to produce the novel geranylgeranyl diphosphate (GGPP) analogue 3-phenyl-3-desmethylgeranylgeranyl diphosphate (3-PhGGPP, ). Our previously developed organocuprate coupling protocol has been used to introduce the cyclopropyl and tert-butyl moieties into the 3-position of vinyl triflate. The four GGPP analogues 3-vinyl-3-desmethylgeranylgeranyl diphosphate (3-vGGPP, ), 3-cyclopropyl-3-desmethylgeranylgeranyl diphosphate (3-cpGGPP, ), 3-tert-butyl-3-desmethyl-geranylgeranyl diphosphate (3-tbGGPP, ), and were then evaluated as potential inhibitors of recombinant yeast protein-geranylgeranyl transferase I (PGGTase I). The potential mechanism-based inhibitors 3-vGGPP and 3-cpGGPP did not exhibit time-dependent inactivation of PGGTase I. Instead, both analogues were alternative substrates, in accord with the interaction of the corresponding farnesyl analogues 3-vFPP and 3-cpFPP with PFTase. The tert-butyl and phenyl analogues were not substrates, but were instead competitive inhibitors of PGGTase I. Note that all four of the GGPP analogues were bound less tightly by the enzyme than the natural substrate, in contrast to the behavior of the 3-substituted FPP analogues.

Alkyl and Aryl Transferases↗
PubMed1998

Properties and functions of the thiamin diphosphate dependent enzyme transketolase.

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Amino Acid Sequence↗
PubMed2003

Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase.

Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.

Alkyl and Aryl Transferases↗
PubMed2006

Hydrophilic anilinogeranyl diphosphate prenyl analogues are Ras function inhibitors.

Sequential processing of H-Ras by protein farnesyl transferase (FTase), Ras converting enzyme (Rce1), and protein-S-isoprenylcysteine O-methyltransferase (Icmt) to give H-Ras C-terminal farnesyl-S-cysteine methyl ester is required for appropriate H-Ras membrane localization and function, including activation of the mitogen-activated protein kinase (MAPK) cascade. We employed a Xenopus laevis oocyte whole-cell model system to examine whether anilinogeranyl diphosphate analogues of similar shape and size, but with a hydrophobicity different from that of the FTase substrate farnesyl diphosphate (FPP), could ablate biological function of H-Ras. Analysis of oocyte maturation kinetics following microinjection of in vitro analogue-modified H-Ras into isoprenoid-depleted oocytes revealed that analogues with a hydrophobicity near that of FPP supported H-Ras biological function, while the analogues p-nitroanilinogeranyl diphosphate (p-NO2-AGPP), p-cyanoanilinogeranyl diphosphate (p-CN-AGPP), and isoxazolaminogeranyl diphosphate (Isox-GPP) with hydrophobicities 2-5 orders of magnitude lower than that of FPP did not. We found that although H-Ras modified with FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP was an efficient substrate for C-terminal postprenylation processing by Rce1 and Icmt, co-injection of H-Ras with analogues p-NO2-AGPP, p-CN-AGPP, or Isox-GPP could not activate MAPK. We propose that H-Ras biological function requires a minimum lipophilicity of the prenyl group to allow important interactions downstream of the C-terminal processed H-Ras protein. The hydrophilic FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP are H-Ras function inhibitors (RFIs) and serve as lead compounds for a unique class of potential anticancer therapeutics.

Alkyl and Aryl Transferases↗
PubMed1999

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.

Alkyl and Aryl Transferases↗
PubMed1973

Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate.

Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution-dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (K(assoc.)=2.3x10(9)), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector.

Allosteric Regulation↗
PubMed1974

Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-diphosphate and methods for characterizing hysteretic transitions.

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.

Adenosine Triphosphate↗
PubMed1999

Molecular cloning and tissue expression of an insect farnesyl diphosphate synthase.

The enzyme farnesyl-diphosphate synthase (FPS, EC2.5.1.1/EC2.5.1.10), which has been shown to play a key role in isoprenoid biosynthesis, catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and di-methylallyl diphosphate. Insects do not synthesize cholesterol de novo, rather farnesyl diphosphate leads to the formation of nonsterol isoprenoids, which are essential for insect development and reproduction. In this paper, we describe the characterization of one FPS from the moth Agrotis ipsilon, the first insect FPS to be reported. An homologous probe was obtained through a nested PCR strategy using degenerate primers designed from the conserved domains of FPS from other organisms. The complete cDNA clone was isolated by PCR screening of a brain cDNA library by using homologous primers deduced from the probe. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 412 amino acids (Mr = 47 170), which shares regions similar to the FPS of other organisms, but exhibits singularities such as an extra N-terminal extension of approximately 70 amino acid residues. Using an RNase protection assay, a protected fragment corresponding to the region encoding the FPS catalytic site was found in brain, ovary, fat body and corpora allata samples, but not in muscle. FPS is overexpressed in the corpora allata, the endocrine gland that produces the juvenile hormones. These hormones are specific to insects and play a crucial role in regulating insect physiology.

Alkyl and Aryl Transferases↗
PubMed2000

Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix. Molecularevolution with alteration in product specificity.

Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates. Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20-C25 diether lipids in addition to C20-C20 diether lipids commonly occurring in archaea [Tachibana, A. (1994) FEBS Lett. 341, 291-294]. Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25-C25 diether lipids, not the usual C20-containing lipids [Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y. & Koga, Y. (1999) Biochim. Biophys. Acta 1436, 426-436]. In this report, we describe the isoloation from A. pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase. The protein encoded by fgs was expressed in Escherichia coli as a glutathione S-transferase fusion protein and produced FGPP as a final product. Phylogenetic analysis of fgs with other prenyltransferases revealed that the short-chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III. fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity.

Alkyl and Aryl Transferases↗
PubMed2006

Structures of eukaryotic ribonucleotide reductase I define gemcitabine diphosphate binding and subunit assembly.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. Crucial for rapidly dividing cells, RNR is a target for cancer therapy. In eukaryotes, RNR comprises a heterooligomer of alpha(2) and beta(2) subunits. Rnr1, the alpha subunit, contains regulatory and catalytic sites; Rnr2, the beta subunit (in yeast, a heterodimer of Rnr2 and Rnr4), houses the diferric-tyrosyl radical crucial for catalysis. Here, we present three x-ray structures of eukaryotic Rnr1 from Saccharomyces cerevisiae: one bound to gemcitabine diphosphate (GemdP), the active metabolite of the mechanism-based chemotherapeutic agent gemcitabine; one with an Rnr2-derived peptide, and one with an Rnr4-derived peptide. Our structures reveal that GemdP binds differently from its analogue, cytidine diphosphate; because of unusual interactions of the geminal fluorines, the ribose and base of GemdP shift substantially, and loop 2, which mediates substrate specificity, adopts different conformations when binding to GemdP and cytidine diphosphate. The Rnr2 and Rnr4 peptides, which block RNR assembly, bind differently from each other but have unique modes of binding not seen in prokaryotic RNR. The Rnr2 peptide adopts a conformation similar to that previously reported from an NMR study for a mouse Rnr2-based peptide. In yeast, the Rnr2 peptide binds at subsites consisting of residues that are highly conserved among yeast, mouse, and human Rnr1s, suggesting that the mode of Rnr1-Rnr2 binding is conserved among eukaryotes. These structures provide new insights into subunit assembly and a framework for structure-based drug design targeting RNR.

Amino Acid Sequence↗
PubMed1996

Arabidopsis thaliana contains two differentially expressed farnesyl-diphosphate synthase genes.

The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain defective in FPS activity (Delourme, D., Lacroute, F., and Karst, F. (1994) Plant Mol. Biol. 26, 1867-1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway.

Alkyl and Aryl Transferases↗
PubMed1996

Compartmentalization of cholesterol biosynthesis. Conversion of mevalonate to farnesyl diphosphate occurs in the peroxisomes.

We have recently demonstrated that mevalonate kinase and farnesyl diphosphate (FPP) synthase are localized predominantly in peroxisomes. This observation raises the question regarding the subcellular localization of the enzymes that catalyze the individual steps in the pathway between mevalonate kinase and FPP synthase (phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase). These enzyme are found in the 100,000 x g supernatant fraction of cells or tissues and have been considered to be cytoplasmic proteins. In the current studies, we show that the activities of mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase are equal in extracts prepared from intact cells and selectively permeabilized cells, which lack cytosolic enzymes. We also demonstrate structure-linked latency of phosphomevalonate kinase and mevalonate diphosphate decarboxylase that is consistent with a peroxisomal localization of these enzymes. Finally, we show that cholesterol biosynthesis from mevalonate can occur in selectively permeabilized cells lacking cytosolic components. These results suggest that the peroxisome is the major site of the synthesis of FPP from mevalonate, since all of the cholestrogenic enzymes involved in this conversion are localized in the peroxisome.

Alkyl and Aryl Transferases↗
PubMed1996

Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate.

The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q). It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G. C., Machado, R. S., and Guest, J. R. (1992) Biochem. J. 287, 611-619). In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate. All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM. Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants. Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the activation lag phase as does the complex. In the parental complexes two distinct lag phases could be resolved, the duration of both decreases with increasing ThDP concentration. A mechanism that is consistent with all of the kinetic data on the parental complexes involves rapid equilibration of the first ThDP with the E1 dimer, followed by a slow conformational equilibration, that in turn is followed by slow addition of the second ThDP to form the fully activated dimer. When the diphosphate site is badly impaired, the binding affinity is very much reduced, this perhaps eliminates the slow step leading to the activated dimer form of the E1.

Acetyltransferases↗
PubMed2003

Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wild-type, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1,600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed1997

Effects of random mutagenesis in a putative substrate-binding domain of geranylgeranyl diphosphate synthase upon intermediate formation and substrate specificity.

Archaeal geranylgeranyl diphosphate (GGPP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce GGPP with significant amounts of intermediates. To obtain information about the amino acids involved in the condensation and the release of intermediates, we randomly mutagenized two proximal regions, I and II, of the Sulfolobus acidocaldarius GGPP synthase gene and created two degenerate libraries, I and II, respectively. Regions I and II correspond to amino acid residues 170-173 and 166-168, respectively. The prenyltransferase activities of about 200 clones were analyzed using the in vivo red-white system and the conventional in vitro assay. Although, in library I, no mutated enzymes that failed to catalyze the formation of GGPP were found, as assayed with the red-white system, almost all the mutated enzymes exhibited weak GGPP synthesis activity, and many produced large amounts of intermediates. The formation of intermediates increased as the concentration of IPP was decreased or as the concentration of the allylic substrate was increased. These phenomena can be regarded as a reflection of the increased K(m) for IPP and the decreased affinity for products including intermediates. On the other hand, no mutants from library II showed such changes. These results suggest that the region from 170 to 173 is concerned in the recognition of both IPP and allylic diphosphates, and that the change in responsiveness to prenyl diphosphates causes a change in intermediate formation.

Alkyl and Aryl Transferases↗
PubMed2000

Significance of Asn-77 and Trp-78 in the catalytic function of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26.

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.

Alkyl and Aryl Transferases↗