The product chain length determination mechanism of prenyltr
The product chain-length determination mechanism of -prenyltransferases has not yet been elucidated, although mutational analyses of highly conserved residues and of characteristic amino azd mg residues in each subfamily of -prenyltransferases have enabled the understanding of the basic catalytic mechanism of those enzymes , , , . Based on the chain length of their products, -prenyltransferases have been classified into three subfamilies, namely, short-, medium-, and long-chain -prenyltransferases, which yield C, C, and C products, respectively . The enzymes of the three groups are thought to possess similar structures and catalytic mechanisms as their amino acid sequences show a high degree of similarity , . X-ray crystallographic analysis of the medium-chain enzyme undecaprenyl diphosphate synthase (UPS) from B-P 26 and revealed that these homodimeric enzymes have a large hydrophobic cleft on the molecular surface of each subunit , . The elongating prenyl chain of the product may stretch along this cleft during enzymatic condensation reactions. Kharel et al. identified some of the characteristic amino acid residues that differ among various -prenyltransferases and examined the function of these residues by the introduction of mutations in B-P 26 UPS . In the long-chain -prenyltransferases, characteristic amino acid insertions were found at the side-wall of the cleft, and it was suggested that these insertions participate in the determination of chain length by controlling the direction of prenyl chain elongation during the catalysis. On the other hand, in the short-chain -prenyltransferase, characteristic amino acid residues found in proximity to the substrate binding site appeared to participate in the determination of chain length of the product by blocking further chain elongation. In the present investigation, mutational studies of the ,-farnesyl diphosphate synthase, Rv1086, from , were performed to help elucidate the mechanism of product chain-length determination of short-chain -prenyltransferases. This enzyme catalyzes the condensation of one molecule of IPP with geranyl diphosphate (GPP) to yield ,-farnesyl diphosphate (,-FPP), and is the only short-chain -prenyltransferase identified thus far. -Prenyltransferases have five conserved regions that are thought to be involved in catalysis . Therefore, mutations were generated in some of these conserved regions. The mutations affected not only the products synthesized by the enzyme but also the specificity for allylic substrates. Data from these studies and mutational studies using B-P 26 UPS indicate that Leu84 plays an important role in the mechanism of product chain-length determination of short- and medium-chain -prenyltransferases. Materials and methods Materials. Precoated reversed-phase TLC plates, namely, LKC-18F, were purchased from Whatman, UK. Non-labeled IPP, GPP, and E,E-FPP were synthesized, as reported . [1-14C]IPP was purchased from GE Healthcare, USA. All other chemicals were of analytical grade. General procedures. Restriction enzyme digestions, transformations, and other standard molecular biology techniques were carried out as described by Sambrook et al. . Plasmid construction and site-directed mutagenesis of Rv1086. For Rv1086 expression, the corresponding gene was amplified by PCR with the following primers: Rv1086UP, 5′-GTACATATGGAGATCATCCCGCCGCGGCTC-3′; and, Rv1086RP, 5′-ACCCTCGAGTGCAACATAGGCGTCC-3′. The sequences corresponding to the NdeI and XhoI sites, which were used in subsequent experiments, are underlined in the primer sequences above in that order. The amplified fragment was cleaved with restriction enzymes and then inserted into an NdeI/XhoI-treated pET-28a vector (Novagen, USA) to construct pET-Rv1086. For construction of +EKE, a Gene Editor in vitro Site-Directed Mutagenesis System (Promega, USA) was used, according to the manufacturer’s protocols. The single stranded rv1086 gene used as a template in the mutagenesis reaction was prepared by infection of E. coli JM109 cells (TaKaRa, Japan) harboring pET-Rv1086 with R408 helper phages. For the construction of mutants L84A, L85A, L85F and L90W, a QuikChange Mutagenesis Kit (STRATAGENE, USA) was used, according to the manufacturer’s protocols. The pET-Rv1086 was used as a template for the construction of mutants. Mutagenic oligonucleotides designed to produce the desired mutant enzymes were: 5′-GAGATCTGCGCAGAGAAGGAGCCGGCCAACCAC-3′ (for +EKE); 5′-CCACCGTCTACGCGTTGTCCACCGAAAACC-3′ (for L84A); 5′-CCGTCTATCTGGCGAGTACTGAAAACCTGCAGC-3′ (for L85A); 5′-CCGTCTATCTGTTCAGTACTGAAAACCTGCAGC-3′ (for L85F); 5′-CCACCGAAAACTGGCAGCGCGATCC-3′ (for L90W). After the confirmation of the introduced mutation by a DNA sequencing, the constructed plasmids were cleaved with the restriction enzymes NdeI/XhoI, which were then inserted into NdeI/XhoI-treated pET-15b vector (Novagen, USA).