A novel geranylgeranyl reductase from the methanogenic archaeon Methanosarcina acetivorans displays unique regiospecificity

Abstract

Saturation of a prenyl group to various levels is a frequently observed modification of isoprenoids. The members of the geranylgeranyl reductase family, however, are the only known enzymes responsible for such reductive modifications in archaea. A methanogenic archaeon, Methanosarcina acetivorans, has proteins homologous to phytoene desaturase CrtI, which is the carotenogenic enzyme that catalyzes oxidation/isomerization of phytoene to lycopene, but their function in carotenogenesis is unlikely in a methanogen that does not produce carotenoids. In the present study, we identified one of the homologues, MA1492, as a new type of archaeal geranylgeranyl reductase that is not homologous to known geranylgeranyl reductases. The expression of MA1492 in Escherichia coli cells, which were genetically modified to produce unsaturated archaeal‐type lipids, led to the production of partially saturated lipid derivatives. Furthermore, we analyzed the substrate specificity of recombinant MA1492 via in vitro assays. The LC‐MS, or radio‐TLC, analysis of the reaction products showed that the enzyme was definitely specific to compounds containing C20 geranylgeranyl groups and reduced only one of four double bonds in a geranylgeranyl chain. The GC‐MS analysis of the product from geranylgeraniol confirmed that the reduction selectively occurred on the ω‐terminal double bond. The available crystallographic structure of an orthologue enzyme may explain the reaction mechanism that achieves the substrate specificity and regiospecificity. Database Microbial Genome Database (http://mbgd.genome.ad.jp/), EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle)

A cis‐prenyltransferase from Methanosarcina acetivorans catalyzes both head‐to‐tail and nonhead‐to‐tail prenyl condensation

“…The chain lengths of the products were, however, still shorter than those of the glycosyl carrier lipids from M. acetivorans. As mentioned in our previous article [18], we detected undecaprenol and dihydroundecaprenol (although the position of the double bond reduction was unclear) via LC-MS analysis of the lipid that was extracted from M. acetivorans and then treated with phosphatase (Fig. 5).…”

“…Total lipids were extracted via a method established by Bligh and Dyer [30] from~3 g of M. acetivorans cells cultivated as described elsewhere [18]. The cells were suspended in 15 mL of water, and 70 mL of methanol and 37.5 mL of chloroform were added.…”

“…acidocaldarius, respectively, as described elsewhere [18]. The assay mixture typically contained, in a total volume of 200 lL,~0.15 nmol of the 14 C-labeled allylic prenyl diphosphate (FPP or GGPP), 0.15 nmol of nonlabeled allylic prenyl diphosphate (DMAPP, GPP, FPP, or GGPP), 20 lmol MOPS-NaOH, pH7.0, 1 lmol MgCl 2 , and 30-400 pmol of purified MA1831 fused with the N-terminal tag.…”

A phytoene desaturase homolog gene from the methanogenic archaeon Methanosarcina acetivorans is responsible for hydroxyarchaeol biosynthesis

“…An E. coli Top10 strain harboring pBAD-ALB4-ma0127 [13] or pBAD-ALB4 [14] was cultivated statically at 37 C for 48 h, in 1.5 L of LuriaeBertani (LB) medium containing 100 mg/ml ampicillin and 0.02%(w/v) L-arabinose. Lipid samples were extracted from the E. coli cells following a method described elsewhere [14].…”

“…LC-ESI-tandem-MS analyses of the lipid samples were performed with an Esquire 3000 ion trap system (Bruker Daltonics, USA) connected to an Agilent 1100 Series HPLC (Agilent Technologies, USA) as described elsewhere [14], with slight modifications as previously reported [13]: the mobile phase was replaced with methanol/2-propanol/100 mg L À1 sodium acetate (7:2:1), and the scanning range was changed to 50e2000 m/z.…”