Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens

Abstract

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO 2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas.

The crystal structure of the malic enzyme fromCandidatusPhytoplasma reveals the minimal structural determinants for a malic enzyme

“…The expression construct was designed to code for an N-terminal hexahistidine (6ÂHis) tag for purification by immobilized metal-affinity chromatography. The recombinant AYWB-ME_Tyr36Ala protein was expressed in Escherichia coli BL21 (DE3) cells previously transformed with pET-AYWB-ME_Tyr36Ala, and the purification protocol was similar to that used for the wild-type protein (Saigo et al, 2014).…”

“…Recombinant protein expression and purification of AYWB-ME were performed as described previously (Saigo et al, 2014). Briefly, AYWB-ME was expressed in E. coli BL21 (DE3) cells and purified by Ni-NTA affinity chromatography followed by size-exclusion chromatography.…”

“…In each organism, different ME isoforms are implicated in diverse and relevant metabolic pathways. For instance, ME is linked to an increase of the photosynthetic yield in maize and sorghum, amongst other plants with economic impact (Saigo et al, 2013;Alvarez et al, 2013); the regulation of stomata opening in tobacco plants (Laporte et al, 2002); plant defence responses (Saigo et al, 2004); oxidative stress and lignin biosynthesis (Casati et al, 1999;Liu et al, 2007); glutamine metabolism in cancerous animal cells (Yang, Lanks et al, 2002); and the metabolism of pathogens such as phytoplasma (Saigo et al, 2014).…”

“…Aster yellows witches'-broom (Candidatus Phytoplasma) ME (AYWB-ME) is an interesting ME with a significantly smaller molecular mass (42 kDa compared with $62 kDa for the well characterized examples), an apparently dimeric quaternary structure and intriguingly distinct functional properties, such as activation by ADP and glutamine, and inhibition by ATP. The molecular mechanisms underlying the pathogenicity of Phytoplasma remain elusive, and it has been suggested that depletion of host resources by the action of AYWB-ME plays a crucial role (Kube et al, 2012;Saigo et al, 2014). AYWB-ME is an excellent model to define the minimal structural determinants of these enzymes and to resolve the question of how a 30% smaller protein scaffold retains full ME activity.…”

The enemy within: phloem‐limited pathogens

“…CPh species are grouped by their 16S rDNA sequence, which remains the sole identifier for many known phytoplasmal pathogens (Bertaccini et al, 2014). CPh pathogens probably use a phytoplasma-specific pathway to generate energy that could play a role in pathogenesis (Bai et al, 2006;Kube et al, 2012;Saigo et al, 2014). They are thought to adhere to cell surfaces, like other mycoplasmas, and may move through the phloem passively, like viruses (Christensen et al, 2005;Lefol et al, 1993;Razin, 1999).…”