Characterization and Functional Identification of a Novel Plant 4,5-Extradiol Dioxygenase Involved in Betalain Pigment Biosynthesis in Portulaca grandiflora

Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism

Comparative Genomics Suggests that the Fungal Pathogen Pneumocystis Is an Obligate Parasite Scavenging Amino Acids from Its Host's Lungs

Pneumocystis jirovecii is a fungus causing severe pneumonia in immuno-compromised patients. Progress in understanding its pathogenicity and epidemiology has been hampered by the lack of a long-term in vitro culture method. Obligate parasitism of this pathogen has been suggested on the basis of various features but remains controversial. We analysed the 7.0 Mb draft genome sequence of the closely related species Pneumocystis carinii infecting rats, which is a well established experimental model of the disease. We predicted 8’085 (redundant) peptides and 14.9% of them were mapped onto the KEGG biochemical pathways. The proteome of the closely related yeast Schizosaccharomyces pombe was used as a control for the annotation procedure (4’974 genes, 14.1% mapped). About two thirds of the mapped peptides of each organism (65.7% and 73.2%, respectively) corresponded to crucial enzymes for the basal metabolism and standard cellular processes. However, the proportion of P. carinii genes relative to those of S. pombe was significantly smaller for the “amino acid metabolism” category of pathways than for all other categories taken together (40 versus 114 against 278 versus 427, P<0.002). Importantly, we identified in P. carinii only 2 enzymes specifically dedicated to the synthesis of the 20 standard amino acids. By contrast all the 54 enzymes dedicated to this synthesis reported in the KEGG atlas for S. pombe were detected upon reannotation of S. pombe proteome (2 versus 54 against 278 versus 427, P<0.0001). This finding strongly suggests that species of the genus Pneumocystis are scavenging amino acids from their host's lung environment. Consequently, they would have no form able to live independently from another organism, and these parasites would be obligate in addition to being opportunistic. These findings have implications for the management of patients susceptible to P. jirovecii infection given that the only source of infection would be other humans

Some features of free-living microorganisms and obligate parasites.

<p>Some features of free-living microorganisms and obligate parasites.</p

Number of enzymes dedicated to the biosyntheses of amino acids identified in <i>P. carinii</i> and <i>S. pombe</i>^a.

a<p>The reference gene numbers of <i>S. pombe</i> are those obtained from KEGG.</p>b<p>The four enzymes are the same for Ile and Val syntheses.</p>c<p>One of the enzymes is also involved in Ile and Val syntheses.</p

Estimation of the quality of the mapping onto KEGG maps by performing a re-prediction of the annotation of <i>S. pombe</i> proteome through intermediary data set consisting of one, two, three, or 18 fungal proteomes.

<p>The KO - <i>S. pombe</i> association pairs obtained by “blasting” an intermediary data set were evaluated <i>a posteriori</i> as true positive (TP) or false positive (FP) according to the KO - <i>S. pombe</i> mapping which is provided by KEGG. Those missed KO - <i>S. pombe</i> pairs existing in KEGG were taken as false negatives (FN). The overall quality of the obtained mapping can be expressed in terms of precision TP/(TP+FP) and recall TP/(TP+FN).</p

Proteomes investigated for transfer the KEGG annotations of the <i>P. carinii</i> predicted proteome.

<p>Proteomes investigated for transfer the KEGG annotations of the <i>P. carinii</i> predicted proteome.</p