The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction

Abstract

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short‐chain (all‐E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate‐rich motif play a primary role in the product determination mechanism. However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate‐rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate‐rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α‐helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase.

Structure of a Heterotetrameric Geranyl Pyrophosphate Synthase from Mint (Mentha piperita) Reveals Intersubunit Regulation

“…Based on structure and sequence comparisons with plant GGPPSs, the two-chamber architecture of LSU in the Mp GPPS structure provides additional cavities (CP hole and EC) for accommodating the longer product of C 20 -GGPP ( Figure 5; see Supplemental Figure 12 online). Our proposed two-chamber mechanism in which the product chain length is regulated by intersubunit interaction is distinct from the previous single-chamber molecular ruler mechanism for wellstudied homomeric PTSs, which use bulky residues to serve as flooring at the bottom of each enzyme active site to block further product chain elongation (Ohnuma et al, 1996;Tarshis et al, 1996;Hemmi et al, 2003;Guo et al, 2004aGuo et al, , 2004bSun et al, 2005;Chang et al, 2006;Noike et al, 2008). When the elongation barrier is removed in the active site of these enzymes by substituting the bulky residue with a smaller one, longer chain length products are generated.…”

“…The function and structure of homomeric PTSs, such as FPPS and GGPPS, have been well studied (Ohnuma et al, 1996;Tarshis et al, 1996;Hemmi et al, 2003;Hosfield et al, 2004;Chang et al, 2006;Gabelli et al, 2006;Kavanagh et al, 2006aKavanagh et al, , 2006bKloer et al, 2006;Rondeau et al, 2006;Guo et al, 2007;Noike et al, 2008). The sequences of different enzymes generally contain <30% conserved amino acids (see Supplemental Figure 1 online).…”

“…These motifs are important for substrate and cofactor binding. Previous studies suggest the existence of a molecular ruler mechanism whereby the contour (shape and size) of the catalytic cavity (tunnel) of PTSs determines the product specificity and substrate selectivity (Ohnuma et al, 1996;Tarshis et al, 1996;Hemmi et al, 2003;Guo et al, 2004aGuo et al, , 2004bSun et al, 2005;Chang et al, 2006;Kavanagh et al, 2006a;Kloer et al, 2006;Thulasiram et al, 2007;Noike et al, 2008).…”

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

“…Mutational and crystallographic analyses of homodimeric type II GGPPs (C 20 synthesis) from Pantoea ananatis and from Sinapis alba (SaGGPPs) (PDB code 2J1P), respectively, have suggested that the bottoms of the clefts were located at the subunit interface (15,34) (Fig. 5C).…”

“…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).…”