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ABSTRACT The plant shikimate pathway directs a significant portion of photosynthetically assimilated carbon into the downstream biosynthetic pathways of aromatic amino acids (AAA) and aromatic natural products. 3‐Deoxy‐d‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase (hereafter DHS) catalyzes the first step of the shikimate pathway, playing a critical role in controlling the carbon flux from central carbon metabolism into the AAA biosynthesis. Previous biochemical studies suggested the presence of manganese‐ and cobalt‐dependent DHS enzymes (DHS‐Mn and DHS‐Co, respectively) in various plant species. Unlike well‐studied DHS‐Mn, however, the identity of DHS‐Co is still unknown. Here, we show that all three DHS isoforms ofArabidopsis thalianaexhibit both DHS‐Mn and DHS‐Co activities in vitro. A phylogenetic analysis of various DHS orthologs and related sequences showed that Arabidopsis 3‐deoxy‐D‐manno‐octulosonate‐8‐phosphate synthase (KDOPS) proteins were closely related to microbial Type I DHSs. Despite their sequence similarity, these Arabidopsis KDOPS proteins showed no DHS activity. Meanwhile, optimization of the DHS assay conditions led to the successful detection of DHS‐Co activity from Arabidopsis DHS recombinant proteins. Compared with DHS‐Mn, DHS‐Co activity displayed the same redox dependency but distinct optimal pH and cofactor sensitivity. Our work provides biochemical evidence that the DHS isoforms of Arabidopsis possess DHS‐Co activity.
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The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light–dark cycle
Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a “fly-casting mechanism.” We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light–dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.
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- PAR ID:
- 10190050
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- Volume:
- 295
- Issue:
- 29
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- 9901 to 9916
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1074/jbc.RA120.012877
