Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K₁) by Spinach Chloroplasts

Abstract

In spinach chloroplasts, 1,4-dihydroxy-2-naphthoate is prenylated by phytyldiphosphate and subsequently methylated by S-adenosylmethionine to form phylloquinol. The site of the prenylation reaction is the chloroplast envelope membrane. [ [I] result in formation of menaquinones of different length of side chain. The principal steps of the synthesis of the naphthoquinone moiety are similar for both plants and bacteria [5 -71. In the present paper we wish to report on the prcnylation of 1,4-dihydroxy-2-naphthoate by phytyldiphosphate and subsequent methylation by S-adenosylmethionine to form phylloquinol in spinach chloroplasts. The site of the prenylation reaction is the chloroplast envelope membrane. 1,4-Dihydroxy-2-naphthoate MATERIALS AND METHODS Radiochenzira1,s and Chemical SynthesisS-[methyl-'4C]Adenosylmethionine (56 Ci/mol) was purchased from Amersham Buchler (Braunschweig, FRG), [U-14C]phytol (80 Ci/mol) from NEN (Dreieich, FRG).[U-'4C]Phytyl diphosphate was prepared according to [8] modified as follows : 0.75 mg (z 200 pCi) of the above labeled phytol dissolved in 25 p1 acetonitrile was added dropwise within 2 h by a micropipette to a 250-pl reaction vessel containing 100 p1 di(triethy1ammonium) phosphate (3.3 % in dry acetonitrile) and 10 pl trichloroacetonitrile. After leaving overnight at 0°C in the dark, phytyl phosphates were precipitated by adding 100 pl acetone plus 5 pl concentrated NH3. After 2 h at -20 "C the precipitate was washed twice with 100 $0.28 M NHJ in methanol to remove phytyl monophosphate. The yield of the diphosphate was 30 pCi. 1,4-Dihydroxy-Znaphthoic acid was prepared from 1 -hydroxy-2-naphthoic acid according to [2], 1,CNaphthoquinones and 2-methyl-3,4-naphthoquinones with phytyl, geranylgeranyl, farnesyl and geranyl side chain, respectively, (2-prenyl-l,4-naphthoquinones and 2-methyl-3-prenyl-1 ,Cnaphthoquinones) were synthesized as described under 'synthesis of menaquinones, general procedure' in [9] aqueous solution of sodium dithionite and was alkalinized by NAOH. After oxidizing the quinol by AgzO in the dark in dry diethylether, the product was chromatographed on a column of silicagel (1.5 x 40 cm) with light petroleum/diethylether (20-10: 1) as solvent. If necessary, the product was rechromatographed with the same system on thin-layer. For chloroplast experiments, prenylated and non-prenylated naphthoquinols were formed by reduction from the corresponding quinones just prior to use: some crystals of NaBH4 were added to a mixture containing 50 p1 of the 1 mM quinone, 2.5 ml methanol, 2.5 ml diethyl ether and 0.5 ml water. After 1 min the quinol was extracted with 10 ml diethyl ether, washed three times with 5 ml water each and quantified photometrically (approximated E243nm 27 500 mol-' 1 ern-', see [9,103). The required amount was evaporated under nitrogen in the incubation vessel of the chloroplast experiments. Isolation of Broken ChloroplastsBroken chloroplasts were prepared from intact chloroplasts isolated according to [ l l , 121; however, the concentration of Mg...

Unexpected roles of plastoglobules (plastid lipid droplets) in vitamin K 1 and E metabolism

“…This was one of the first indications that PG function not only in the storage but also partici-pate in the metabolism of tocopherol [15,20] (Figure 2). The vte1 mutant lacking TC and the vte2 mutant lacking the homogentisate phytyl transferase are both devoid of a-tocopherol.…”

“…The majority of the chloroplast enzymes implicated in the biosynthesis of prenylquinones have been localized at the inner membrane of the chloroplast envelope [13][14][15], with the exception of part of the phylloquinone biosyn-thetic pathway that is compartmentalized in peroxisomes [16,17] (Figure 1). …”

A Plant Locus Essential for Phylloquinone (Vitamin K1) Biosynthesis Originated from a Fusion of Four Eubacterial Genes

“…In plants, relatively little is known about PhQ biosynthesis, although biochemical analysis suggested a metabolic pathway similar to that of bacteria and that the principle reactions occur at the chloroplast envelope membrane (7). The menA homologous Arabidopsis thaliana gene, AtmenA, was recently demonstrated to be essentially required for PhQ biosynthesis and accumulation of PSI.…”

Deficiency in Phylloquinone (Vitamin K1) Methylation Affects Prenyl Quinone Distribution, Photosystem I Abundance, and Anthocyanin Accumulation in the Arabidopsis AtmenG Mutant

“…The At1g23360 Protein Localizes to Chloroplasts-The enzymatic activity of PNQ methyltransferase was previously localized to the envelope membranes of spinach chloroplasts (11). Therefore, it was expected that the At1g23360 protein localizes to chloroplasts.…”

“…Phylloquinone synthesis in plants has been studied by feeding biosynthetic precursors and by enzyme assays in spinach, Capsicum annuum, Euglena gracilis, and Galium cell cultures (11)(12)(13)(14). The first genetic study of phylloquinone synthesis in plants was presented by the isolation of the AtmenA mutant of Arabidopsis carrying a block in DHNA phytyltransferase.…”