Homodimeric Hexaprenyl Pyrophosphate Synthase from the Thermoacidophilic Crenarchaeon Sulfolobus solfataricus Displays Asymmetric Subunit Structures

Abstract

Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-C 30 -hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranylgeranyl pyrophosphate. The crystal structure of the homodimeric C 30 -HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). In both subunits, 10 core helices are arranged about a central active site cavity. Leu164 in the middle of the cavity controls the product chain length. Two protein conformers are observed in the S. solfataricus HexPPs structure, and the major difference between them occurs in the flexible region of residues 84 to 100. Several helices (␣I, ␣J, ␣K, and part of ␣H) and the associated loops have high-temperature factors in one monomer, which may be related to the domain motion that controls the entrance to the active site. Different side chain conformations of Trp136 in two HexPPs subunits result in weaker hydrophobic interactions at the dimer interface, in contrast to the symmetric -stacking interactions of aromatic side chains found in FPPs and OPPs. Finally, the three-conformer switched model may explain the catalytic process for HexPPs.Isoprenoids are among the most diverse and widely distributed natural compounds (17,22). Using the 5-carbon isopentenyl pyrophosphate (IPP) as building blocks, linear isoprenoids are synthesized by a group of prenyltransferases (PTases), which catalyze the multiple-IPP condensation reaction with allylic substrates, i.e., C 5 dimethyl allyl pyrophosphate (DMAPP), C 15 farnesyl pyrophosphate (FPP), or C 20 geranylgeranyl pyrophosphate (GGPP) for chain elongation (13). These PTases have been classified as E-and Z-types, which synthesize products with trans and cis double bonds, respectively. Different enzymes synthesize varying chain length products, which are then utilized as precursors for steroids, carotenoids, quinones, dilichols, prenylated proteins, and archaeal membrane lipids.Trans-PTases are typically classified into three groups based on the chain length of their final products, i.e., short (C 10 to C 25 ), medium (C 30 to C 35 ), and long chain (C 40 to C 50 ) (12, 16). The crystal structures of short-chain farnesyl pyrophosphate synthase (FPPs) and long-chain octaprenyl pyrophosphate synthase (OPPs) have been determined previously (6,10,25). These proteins are homodimers. Each subunit contains several ␣-helices that form a large central active site cavity with two conserved DDXXD motifs, located at the rims of the helices D and H, respectively. On the other hand, the mediumchain trans-PTases, including hexaprenyl pyrophosphate synthase (HexPPs) and heptaprenyl pyrophosphate synthase (HepPPs), are composed of either homodimers or heterodimers. The HexPPs from Sulfolobus solfataricus (23), which catalyzes the condensation of two IPP molecules with GGPP to yield all-trans-C 30 -HexPP, is a homodimer (8). In contrast, HexPPs from Micrococcus luteus...

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

“…The dominant roles of the small subunit seem to consist of stabilizing the functional heterodimeric unit of the enzyme through the large hydrophobic interface and directly regulating the product chain length together with the large catalytic subunit HexB. ) previously reported by Sun et al (11). The estimated volume of chain B is probably different from that holding the ligand because the chain is in "open" conformation to release the product (11).…”

“…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].…”