A highly conserved mechanism for the detoxification and assimilation of the toxic phytoproduct L-azetidine-2-carboxylic acid in Aspergillus nidulans

Plants produce toxic secondary metabolites as defense mechanisms against phytopathogenic microorganisms and predators. L-azetidine-2-carboxylic acid (AZC), a toxic proline analogue produced by members of the Liliaceae and Agavaciae families, is part of such a mechanism. AZC causes a broad range of toxic, inflammatory and degenerative abnormalities in human and animal cells, while it is known that some microorganisms have evolved specialized strategies for AZC resistance. However, the mechanisms underlying these processes are poorly understood. Here, we identify a widespread mechanism for AZC resistance in fungi. We show that the filamentous ascomycete Aspergillus nidulans is able to not only resist AZC toxicity but also utilize it as a nitrogen source via GABA catabolism and the action of the AzhA hydrolase, a member of a large superfamily of detoxifying enzymes, the haloacid dehalogenase-like hydrolase (HAD) superfamily. This detoxification process is further assisted by the NgnA acetyltransferase, orthologue of Mpr1 of Saccharomyces cerevisiae. We additionally show that heterologous expression of AzhA protein can complement the AZC sensitivity of S. cerevisiae. Furthermore, a detailed phylogenetic analysis of AzhA homologues in Fungi, Archaea and Bacteria is provided. Overall, our results unravel a widespread mechanism for AZC resistance among microorganisms, including important human and plant pathogens.

Genome-wide analysis of haloacid dehalogenase genes reveals their function in phosphate starvation responses in rice

The HAD superfamily is named after the halogenated acid dehalogenase found in bacteria, which hydrolyses a diverse range of organic phosphate substrates. Although certain studies have shown the involvement of HAD genes in Pi starvation responses, systematic classification and bioinformatics analysis of the HAD superfamily in plants is lacking. In this study, 41 and 40 HAD genes were identified by genomic searching in rice and Arabidopsis, respectively. According to sequence similarity, these proteins are divided into three major groups and seven subgroups. Conserved motif analysis indicates that the majority of the identified HAD proteins contain phosphatase domains. A further structural analysis showed that HAD proteins have four conserved motifs and specified cap domains. Fewer HAD genes show collinearity relationships in both rice and Arabidopsis, which is consistent with the large variations in the HAD genes. Among the 41 HAD genes of rice, the promoters of 28 genes contain Pi-responsive cis-elements. Mining of transcriptome data and qRT-PCR results showed that at least the expression of 17 HAD genes was induced by Pi starvation in shoots or roots. These HAD proteins are predicted to be involved in intracellular or extracellular Po recycling under Pi stress conditions in plants.

A novel structurally characterized haloacid dehalogenase superfamily phosphatase from Thermococcus thioreducens with diverse substrate specificity

The haloacid dehalogenase (HAD) superfamily is one of the largest known groups of enzymes and the majority of its members catalyze the hydrolysis of phosphoric acid monoesters into a phosphate ion and an alcohol. Despite the fact that sequence similarity between HAD phosphatases is generally very low, the members of the family possess some characteristic features, such as a Rossmann-like fold, HAD signature motifs or the requirement for Mg2+ ion as an obligatory cofactor. This study focuses on a new hypothetical HAD phosphatase from Thermococcus thioreducens. The protein crystallized in space group P21212, with unit-cell parameters a = 66.3, b = 117.0, c = 33.8 Å, and the crystals contained one molecule in the asymmetric unit. The protein structure was determined by X-ray crystallography and was refined to 1.75 Å resolution. The structure revealed a putative active site common to all HAD members. Computational docking into the crystal structure was used to propose substrates of the enzyme. The activity of this thermophilic enzyme towards several of the selected substrates was confirmed at temperatures of 37°C as well as 60°C.

Haloacid Dehalogenase Proteins: Novel Mediators of Metabolic Plasticity in Plasmodium falciparum

Widespread antimalarial drug resistance has prompted the need for new therapeutics and greater understanding of malaria parasite biology. To this end, the isoprenoid biosynthesis inhibitor fosmidomycin has been used to probe the metabolic regulation in the malaria parasite, Plasmodium falciparum. Genetic changes in the haloacid dehalogenase (HAD) superfamily member HAD2 conferred resistance to fosmidomycin, at the cost of decreased fitness. In the absence of fosmidomycin, parasites gained mutations to phosphofructokinase that restored growth and fosmidomycin sensitivity, thus revealing an intriguing example of plasticity in a core glycolytic process. Moreover, this study marks a second report of a HAD superfamily protein-modulating metabolic homeostasis in P falciparum parasites. Haloacid dehalogenase enzymes are distributed across all domains of life and have increasingly been found to influence central carbon metabolism and drug sensitivity in P falciparum. Investigating the mechanisms by which HAD superfamily members modulate metabolism may shed light on how metabolic networks are connected in apicomplexan parasites and other organisms and may guide future therapeutic endeavors.

A preliminary X-ray study of 3-deoxy-D-manno-oct-2-ulosonic acid 8-phosphate phosphatase (YrbI) fromBurkholderia pseudomallei

3-Deoxy-D-manno-oct-2-ulosonic acid 8-phosphate phosphatase (YrbI), the third enzyme in the pathway for the biosynthesis of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO), hydrolyzes KDO 8-phosphate to KDO and inorganic phosphate. YrbI belongs to the haloacid dehalogenase (HAD) superfamily, which is a large family of magnesium-dependent phosphatase/phosphotransferase enzymes. In this study, YrbI fromBurkholderia pseudomallei, the causative agent of melioidosis, has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.25 Å resolution. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 63.7,b= 97.5,c= 98.0 Å. A full structural determination is in progress to elucidate the structure–function relationship of this protein.

Deletion ofPHO13, Encoding Haloacid Dehalogenase Type IIA Phosphatase, Results in Upregulation of the Pentose Phosphate Pathway in Saccharomyces cerevisiae

ABSTRACTThe haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes inSaccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identifiedPHO13to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion ofPHO13(pho13Δ), we investigated the response ofS. cerevisiaetopho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed thatpho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced bypho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus,pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation bypho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function ofS. cerevisiaePho13 and the HAD superfamily enzymes and for developingS. cerevisiaestrains with industrially attractive phenotypes.