Polyprenyl Diphosphate Synthases

111

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C 40 octaprenyl pyrophosphate (OPP), which constitutes the side chain of bacterial ubiquinone or menaquinone. In this study, the first structure of long chain C 40 -OPPs from Thermotoga maritima has been determined to 2.28-Å resolution. OPPs is composed entirely of ␣-helices joined by connecting loops and is arranged with nine core helices around a large central cavity. An elongated hydrophobic tunnel between D and F ␣-helices contains two DDXXD motifs on the top for substrate binding and is occupied at the bottom with two large residues Phe-52 and Phe-132. The products of the mutant F132A OPPs are predominantly C 50 , longer than the C 40 synthesized by the wild-type and F52A mutant OPPs, suggesting that Phe-132 is the key residue for determining the product chain length. Ala-76 and Ser-77 located close to the FPP binding site and Val-73 positioned further down the tunnel were individually mutated to larger amino acids. A76Y and S77F mainly produce C 20 indicating that the mutated large residues in the vicinity of the FPP site limit the substrate chain elongation. Ala-76 is the fifth amino acid upstream from the first DDXXD motif on helix D of OPPs, and its corresponding amino acid in FPPs is Tyr. In contrast, V73Y mutation led to additional accumulation of C 30 intermediate. The new structure of the trans-type OPPs, together with the recently determined cis-type UPPs, significantly extends our understanding on the biosynthesis of long chain polyprenyl molecules.Prenyltransferases catalyze consecutive condensation reactions of isopentenyl pyrophosphate (IPP) 1 with allylic pyrophosphate to generate linear isoprenyl polymers (1-3). Starting from IPP and its isomer dimethylallyl pyrophosphate, the C 15 farnesyl pyrophosphate (FPP) is formed by farnesyl pyrophosphate synthase (FPPs) (4). Using FPP and IPP as substrates, C 40 octaprenyl pyrophosphate (OPP) is synthesized by octaprenyl pyrophosphate synthase (OPPs) via five IPP condensation reactions with FPP (5, 6). This polymer serves as the side chain of bacterial ubiquinone or menaquinone, a component involved in electron transfer for oxidative phosphorylation (7). Previously we have identified an OPPs from hyperthermophilic bacterium Thermotoga maritima (8). Compared with its mesophilic counterpart OPPs in Escherichia coli (9), the thermophilic enzymes shows higher product specificity, higher thermal stability, and lower structural flexibility.During each IPP condensation, a new double bond is formed. Thus the prenyltransferases are classified as cis-and transtype depending on the stereoisomer of the double bond formed (10). Two DDXXD motifs in the amino acid sequences were found in trans-type prenyltransferases. The first motif is responsible for binding with FPP, and the second motif is responsible for IPP binding (11)(12)(13)(14). OPPs is a trans-type enzyme synthesizing the C 40 long chain pro...

Section: Mutant Namementioning

confidence: 99%

“…Prenyltransferases catalyze consecutive condensation reactions of isopentenyl pyrophosphate (IPP) 1 with allylic pyrophosphate to generate linear isoprenyl polymers (1)(2)(3). Starting from IPP and its isomer dimethylallyl pyrophosphate, the C 15 farnesyl pyrophosphate (FPP) is formed by farnesyl pyrophosphate synthase (FPPs) (4).…”

mentioning

confidence: 99%

Crystal Structure of Octaprenyl Pyrophosphate Synthase from Hyperthermophilic Thermotoga maritima and Mechanism of Product Chain Length Determination

Guo

Kuo

Chou

et al. 2004

111

“…Prenyltransferases catalyze consecutive condensation reactions of isopentenyl pyrophosphate (IPP) 1 with allylic pyrophosphate to generate linear isoprenyl polymers. The isoprenylates undergo further modification to form a variety of isoprenoid structures including steroids, terpenes, the side chains of respiratory quinones, carotenoids, natural rubber, the glycosyl carrier lipid, and prenyl proteins (1,2).…”

mentioning

confidence: 99%

Mechanism of Product Chain Length Determination and the Role of a Flexible Loop in Escherichia coliUndecaprenyl-pyrophosphate Synthase Catalysis

Chen

Robinson

et al. 2001

179

The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the ␤A-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four ␤-strands (␤A-␤B-␤D-␤C) and two helices (␣2 and ␣3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C 70 polymer rather than the C 55 polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C 30 intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C 55 product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C 55 product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k cat values. W75A showed an 8-fold increase of the FPP K m value, and 22-33-fold increases in the IPP K m values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release. Prenyltransferases catalyze consecutive condensation reactions of isopentenyl pyrophosphate (IPP)1 with allylic pyrophosphate to generate linear isoprenyl polymers. The isoprenylates undergo further modification to form a variety of isoprenoid structures including steroids, terpenes, the side chains of respiratory quinones, carotenoids, natural rubber, the glycosyl carrier lipid, and prenyl proteins (1, 2). E-and Z-type prenyltransferases synthesize trans and cis double bonds, respectively, through the condensation reactions of IPP (3). Each of the E-type enzymes catalyzes the formation of a product having a specific chain length ranging from C 10 to C 50 (4).Two conserved DDXXD motifs are observed in E-type enzymes (5-7). X-ray structural (8) and site-directed mutagenesis studies (9 -12) of farnesyl-pyrophosphate synthase (FPPs) have shown that the first aspartate-rich motif binds the allylic substrate, whereas the second DDXXD binds IPP via Mg 2ϩ . Mutagenesis studies indicate that the 5th amino acid residue (Phe-77) upstream from the first DDXXD plays a critical role in controlling the chain length of the final product formed in the reaction catalyzed by E-type geranylgeranyl-pyrophosphate synthase from archaebacterium (13). By substituting this large residue with the smaller Ser, product synthesis was shifted from the production of C 20 product to C 25 and C 30 products (14). Double (F77...

“…Prenyltransferases, also referred to as prenyl diphosphate synthases, are enzymes catalyzing the sequential condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to produce linear prenyl diphosphates in the biosynthetic pathway of isoprenoid compounds, most of which are essential components of the cellular machinery, such as cholesterol, carotenoids, prenyl quinones, prenyl proteins, and dolichols. Although these condensation reactions are similar in terms of chemical mechanism, there are a number of enzymes having different specificities with respect to the chain length and double-bond stereochemistry of its final product.…”

mentioning

confidence: 99%

“…Although these condensation reactions are similar in terms of chemical mechanism, there are a number of enzymes having different specificities with respect to the chain length and double-bond stereochemistry of its final product. These enzymes can be classified into two major subgroups (cis and trans types) according to the geometry of the products and are extremely interesting from an enzymological viewpoint in which the reactions are regulated to proceed consecutively and terminate precisely at definite chain lengths depending on the specificities of individual enzymes (1,2). To date, the structural genes for many types of trans-prenyltransferases have been cloned and characterized.…”

mentioning

confidence: 99%

Identification of Significant Residues for Homoallylic Substrate Binding of Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase

Kharel

Zhang

Fujihashi

et al. 2001

Self Cite

The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Biol. Chem. 272, 19476 -19481). To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the con- Prenyltransferases, also referred to as prenyl diphosphate synthases, are enzymes catalyzing the sequential condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to produce linear prenyl diphosphates in the biosynthetic pathway of isoprenoid compounds, most of which are essential components of the cellular machinery, such as cholesterol, carotenoids, prenyl quinones, prenyl proteins, and dolichols. Although these condensation reactions are similar in terms of chemical mechanism, there are a number of enzymes having different specificities with respect to the chain length and double-bond stereochemistry of its final product. These enzymes can be classified into two major subgroups (cis and trans types) according to the geometry of the products and are extremely interesting from an enzymological viewpoint in which the reactions are regulated to proceed consecutively and terminate precisely at definite chain lengths depending on the specificities of individual enzymes (1, 2). To date, the structural genes for many types of trans-prenyltransferases have been cloned and characterized. Multiple alignments of the deduced amino acid sequences of these transprenyltransferases showed the presence of several conserved regions in the primary structures (3, 4). The conserved regions include two characteristic aspartate-rich motifs (DDXXD), which have been shown to be essential for the catalytic function as well as substrate binding by site-directed mutational analysis (5-8). Tarshis et al. (9) determined the crystal structure of chicken FPP synthase, in which most of the conserved regions are found in a large central cavity. and Tarshis et al. (15) have intensively investigated the product chain length determination mechanisms of short chain transprenyltransferases such as FPP synthase from Bacillus stearothermophilus or chicken and geranylgeranyl diphosphate (GGPP) synthase from Sulfolobus acidocaldarius.On the other hand, cis-prenyltransferases catalyze cis-prenyl chain elongation to produce prenyl diphosphates with E,Zmixed stereochemistry. In bacteria, sequential cis addition of IPP onto FPP as an allylic primer to give undecaprenyl diphosphate (UPP, C 55 ) is catalyzed by UPP synthase. UPP is the direct precursor of glycosyl carrier lipid in the biosynthesis of bacterial cell wall polysaccharide components such as peptidoglycan and lipopolysaccharide. In eukaryotic cells, dehydrodolichyl diphosphate synthase responsible for cis-prenyl chain elongation catalyzes much longer chain elongation than does the bacterial enzyme. Dehydrodolichyl diphosphate is an essential precursor of carbohydrate carrier lipid in the biosynthesis of N-linked glycoprotein or glycosylphosphatidylinositolan...