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

Abstract

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...

Crystal Structure of Heterodimeric Hexaprenyl Diphosphate Synthase from Micrococcus luteus B-P 26 Reveals That the Small Subunit Is Directly Involved in the Product Chain Length Regulation

“…Homodimeric octaprenyl diphosphate synthase (OPPs; C 40 synthesis) from Thermotoga maritima (TmOPPs) has a "tunnel" connecting the two condensation reaction sites on the two subunits (Fig. 5D) in addition to the two deep clefts parallel to the bundle of the helices (PDB code 1V4E) (9,33). The cooperative regulation of the product chain length might be widely seen in both homo-and heterooligomeric trans-prenyltransferases synthesizing medium or long prenyl chain.…”

“…The crystal structure of homodimeric Sc-GGPPs revealed that Tyr and His residues located at the bottom of the cleft contacted the -end of the product GGPP and obstructed further chain elongation (PDB code 2E8V) (12). It is widely believed that each subunit of the homodimer in homooligomeric enzymes independently synthesizes the elongated product with a desired chain length (9,11,12,33,37,43). In the case of heterotetrameric Mp-GPPs, the condensation reaction and the C 10 chain length control are essentially dominated only by the large subunit LSU (59).…”

“…On the other hand, the interior part of the cleft accommodates the hydrophobic tail of the reaction product. The size of the cleft is strongly involved in the regulation of the final product chain length (9,11,12,26,(33)(34)(35)(36)(37)(38)(39). The crystal structure of GGPP synthase from Saccharomyces cerevisiae (Sc-GGPPs) with its final product GGPP revealed that the hydrophobic tail of the product just fits the cleft in both of the two homodimeric subunits (18).…”

“…The cleft shape and size are also the key factor to develop the inhibitors of these enzymes, such as bisphosphonates, to medicate osteoporosis, Paget disease, and hypercalcemia (13, 14, 16 -20, 40 -42). It should be noted that almost all mutational investigations to extend the product chain length are designed to make the cleft deeper along with the antiparallel ␣-helices because the hydrophobic chain is commonly believed to extend along the helices (9,11,12,33,37,43).…”

Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1)

“…In our previous studies, mercury can bind to free Cys residues very easily and the phase problem can be easily solved by using at least two mercury datasets by MIR [18][19][20][21]. The mercury atom binding sites were located using AutoSol wizard of PHENIX using 2.4 Å resolution cutoff [22].…”