Catalytic reactions of the homogentisate prenyl transferase involved in plastoquinone-9 biosynthesis

Abstract

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10-20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K(m) value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.

FIGURE 1.

Aromatic prenyltransferase reaction in PQ-9 biosynthesis (A) and structure of the herbicide haloxydine (B). PPi, diphosphate.

FIGURE 2.

Inhibition of HSTs by haloxydine. Chloroform extracts from assays with C. reinhardtii HST and native HST in chloroplast envelope membranes of spinach were analyzed via TLC using dichloromethane (A) and toluene:isoamyl alcohol:acetic acid (80:40:3) (B) as mobile phase. The enzymes were assayed in the absence (− I) or presence (+ I) of 0.5 mm haloxydine in standard reaction mixtures as described under “Experimental Procedures.” Control assays lacked prenyl donor. * marks the origin; 1 and 2, MFBQ quinol and quinone form, respectively.

FIGURE 3.

TLC analyses of C. reinhardtii HST products from assays with various prenyl donors. Assays were conducted in the presence of 0.1 mm n-dodecyl β-d-maltoside with the exception of sample SPP_B that contained 0.2 mm detergent in the reaction mixture. The chloroform soluble products were separated by TLC using dichloromethane (A) or toluene:isoamyl alcohol:acetic acid (80:40:3) (B) as mobile phase. Besides the expected 2-methyl-6-prenyl-1,4-benzoquinols (marked by 1, 3, 5, and 7) and the respective quinones (marked by 2, 4, 6, and 8), chloroform extracts of assays with prenyl donors up to a chain length of C₂₀ contained mainly so far unknown prenylated polar products. * indicates the origin; PPi, diphosphate. C, the structures for the 2-methyl-6-prenyl-1,4-benzoquinols (upper) and the more polar 6-prenyl-1,4-benzoquinol-2-methylcarboxylates (lower) are given with n = 2, n = 3, n = 4, and n = 9 for the respective GPP-, FPP-, GGPP-, and SPP-derived products.

FIGURE 4.

Kinetics of inhibition of C. reinhardtii HST by haloxydine. The effects of haloxydine on HST-catalyzed MFBQ formation at 200 μm FPP (A) and at 100 μm homogentisate (B) as well as on total product formation at 200 μm FPP (C) and 100 μm homogentisate (D) are shown.

FIGURE 5.

Inhibition of C. reinhardtii HST by haloxydine and a bisphosphonate. A, the inhibitor concentrations are indicated. Total product (FBQC, light gray bars) and MFBQ (dark gray bars) formation is affected to different extents depending on the inhibitor. Data represent mean values and standard deviations of quadruple estimations. The structure of the bisphosphonate is shown in B. Note that this compound was an S enantiomer; the R enantiomer also inhibited similarly.

FIGURE 6.

Suggested pathways for HST-catalyzed farnesylation of homogentisate. In Enzyme Cycle A, homogentisate is decarboxylated to 2-methylquinol prior to prenyl transfer, whereas in the major pathway, Enzyme Cycle B, prenylation occurs without decarboxylation. E, free HST enzyme; PP, pyrophosphate.