Potential roles of YCF54 and ferredoxin‐NADPH reductase for magnesium protoporphyrin monomethylester cyclase

Abstract

Chlorophyll is synthesized from activated glutamate in the tetrapyrrole biosynthesis pathway through at least 20 different enzymatic reactions. Among these, the MgProto monomethylester (MgProtoME) cyclase catalyzes the formation of a fifth isocyclic ring to tetrapyrroles to form protochlorophyllide. The enzyme consists of two proteins. The CHL27 protein is proposed to be the catalytic component, while LCAA/YCF54 likely acts as a scaffolding factor. In comparison to other reactions of chlorophyll biosynthesis, this enzymatic step lacks clear elucidation and it is hardly understood, how electrons are delivered for the NADPH-dependent cyclization reaction. The present study intends to elucidate more precisely the role of LCAA/YCF54. Transgenic Arabidopsis lines with inactivated and overexpressed YCF54 reveal the mutual stability of YCF54 and CHL27. Among the YCF54-interacting proteins, the plastidal ferredoxin-NADPH reductase (FNR) was identified. We showed in N. tabacum and A. thaliana that a deficit of FNR1 or YCF54 caused MgProtoME accumulation, the substrate of the cyclase, and destabilization of the cyclase subunits. It is proposed that FNR serves as a potential donor for electrons required in the cyclase reaction and connects chlorophyll synthesis with photosynthetic activity.

Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54

“…As a cellular electron currency, NADPH is utilized to reduce many redox-active components, and the biochemical fractions used for in vitro cyclase assays probably contained some of these redox-active components, one of which could be the direct electron donor to the diiron center of cyclase. Herbst et al [20] recently proposed that the plastidal ferredoxin-NADPH reductase (FNR) could be involved in reducing the cyclase based on the interaction between FNR1 and YCF54, and the disturbed cyclase activity that resulted from FNR1 deficiency. In oxygenic phototrophs FNR utilizes the electrons from photosynthetically reduced ferredoxin (Fd) to produce NADPH, and when there is a shortage of reduced Fd, such as in the dark, the enzyme can also catalyze the reverse reaction.…”

“…Our work further confirms that Ycf54 is an authentic cyclase subunit in oxygenic phototrophs. In their native hosts, Ycf54 is required for the accumulation of AcsF as demonstrated in Synechocystis [5,18,19], tobacco [6] and Arabidopsis [20] and its interaction with AcsF, mediated by the conserved R82 residue that forms part of a positively charged patch on Ycf54, is required for optimal PChlide formation as shown in Synechocystis [19]. The dependency of AcsF accumulation on Ycf54 was also found for the Arabidopsis enzyme when heterologously expressed in E. coli, but not for the Synechocystis cyclase as indicated by the unaffected level of CycI in the absence of Ycf54 (Figure 8).…”

“…In the search for the 'missing' soluble component, Ycf54/LCAA was identified as an auxiliary cyclase subunit from Synechocystis by in vivo pulldown experiments using FLAG-tagged CycI and CycII as bait [5], and from tobacco by screening antisense plants with Chl deficiency [6]. The key role of Ycf54 proteins in cyclase function is exemplified by its ubiquity in oxygenic phototrophs and the severe phenotypes exhibited by knockout mutants including impaired AcsF protein levels, accumulation of MgPME, and lowered amounts of DV PChlide a and Chl [5,6,[18][19][20][21].…”

The cloning and characterization of hypersensitive to salt stress mutant, affected in quinolinate synthase, highlights the involvement of NAD in stress‐induced accumulation of ABA and proline

“…Chlorophyll contents were slightly decreased in the hss plants under control conditions but the complementation with QS gene restored it to WT levels, suggesting that NAD might affect chlorophyll synthesis. Perhaps it is because NADP(H) or NAD(H) was also required for chlorophyll synthesis and the conversion of chlorophyll a to chlorophyll b (Scheumann et al , ; Folly and Engel, ; Herbst et al , ). It is conceivable that chlorophyll synthesis in salt‐treated hss mutant should be further affected by even lower contents of NADH and NADPH under salt stress.…”

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

“…The reverse reaction can also occur [ 32 ]. Recently, it was proposed that FNR could act as a direct electron donor to the cyclase or create an NADPH-rich environment at the cyclase complex [ 33 ]. Our in vitro assay requires Fd, FNR, and NADPH for activity, which suggests that Fd is the true donor of electrons to XanL as cyclase activity was not obtained with only NADPH added or with NADPH in combination with FNR.…”