Analysis of histidine-tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry.

RATIONALE: Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). METHODS: A protein standard, His-Ubq, and two recombinant proteins, His-SHAN and His-CS, expressed in Escherichia coli were immobilized on two immobilized metal affinity systems, Cu-nitriloacetic acid (Cu-NTA) and Ni-NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96-well plate form factor, or analyzed directly from immobilized metal affinity-coated microscope slides by DESI-MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. RESULTS: Small proteins (His-Ubq) and medium proteins (His-SAHN) could readily be detected from 96-well plates by direct infusion ESI, or from microscope slides by DESI-MS after purification on surface from clarified E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu-NTA and Ni-NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His-SAHN and the methylation product of His-CS (theobromine to caffeine) were detected. CONCLUSIONS: The immobilization, purification, release and detection of His-tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI-MS or ambient DESI-MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

Convergent Biochemical Pathways for Xanthine Alkaloid Production in Plants Evolved from Ancestral Enzymes with Different Catalytic Properties.

Convergent evolution is widespread but the extent to which common ancestral conditions are necessary to facilitate the independent acquisition of similar traits remains unclear. In order to better understand how ancestral biosynthetic catalytic capabilities might lead to convergent evolution of similar modern-day biochemical pathways, we resurrected ancient enzymes of the caffeine synthase (CS) methyltransferases that are responsible for theobromine and caffeine production in flowering plants. Ancestral CS enzymes of Theobroma, Paullinia, and Camellia exhibited similar substrate preferences but these resulted in the formation of different sets of products. From these ancestral enzymes, descendants with similar substrate preference and product formation independently evolved after gene duplication events in Theobroma and Paullinia. Thus, it appears that the convergent modern-day pathways likely originated from ancestral pathways with different inferred flux. Subsequently, the modern-day enzymes originated independently via gene duplication and their convergent catalytic characteristics evolved to partition the multiple ancestral activities by different mutations that occurred in homologous regions of the ancestral proteins. These results show that even when modern-day pathways and recruited genes are similar, the antecedent conditions may be distinctive such that different evolutionary steps are required to generate convergence.

Novel genetic code and record-setting AT-richness in the highly reduced plastid genome of the holoparasitic plant Balanophora.

Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The two Balanophora plastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thus Balanophora plastids must import all tRNAs needed for translation. Balanophora plastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of all cis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, the Balanophora genomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes in Balanophora consist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used by Balanophora plastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, the Balanophora plastome must be functional because all examined genes are transcribed, its only intron is correctly trans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes.

Convergent evolution is a process that has occurred throughout the tree of life, but the historical genetic and biochemical context promoting the repeated independent origins of a trait is rarely understood. The well-known stimulant caffeine, and its xanthine alkaloid precursors, has evolved multiple times in flowering plant history for various roles in plant defense and pollination. We have shown that convergent caffeine production, surprisingly, has evolved by two previously unknown biochemical pathways in chocolate, citrus, and guaraná plants using either caffeine synthase- or xanthine methyltransferase-like enzymes. However, the pathway and enzyme lineage used by any given plant species is not predictable from phylogenetic relatedness alone. Ancestral sequence resurrection reveals that this convergence was facilitated by co-option of genes maintained over 100 million y for alternative biochemical roles. The ancient enzymes of the Citrus lineage were exapted for reactions currently used for various steps of caffeine biosynthesis and required very few mutations to acquire modern-day enzymatic characteristics, allowing for the evolution of a complete pathway. Future studies aimed at manipulating caffeine content of plants will require the use of different approaches given the metabolic and genetic diversity revealed by this study.

Miniaturized mitogenome of the parasitic plant Viscum scurruloideum is extremely divergent and dynamic and has lost all nad genes.

Despite the enormous diversity among parasitic angiosperms in form and structure, life-history strategies, and plastid genomes, little is known about the diversity of their mitogenomes. We report the sequence of the wonderfully bizarre mitogenome of the hemiparasitic aerial mistletoe Viscum scurruloideum. This genome is only 66 kb in size, making it the smallest known angiosperm mitogenome by a factor of more than three and the smallest land plant mitogenome. Accompanying this size reduction is exceptional reduction of gene content. Much of this reduction arises from the unexpected loss of respiratory complex I (NADH dehydrogenase), universally present in all 300+ other angiosperms examined, where it is encoded by nine mitochondrial and many nuclear nad genes. Loss of complex I in a multicellular organism is unprecedented. We explore the potential relationship between this loss in Viscum and its parasitic lifestyle. Despite its small size, the Viscum mitogenome is unusually rich in recombinationally active repeats, possessing unparalleled levels of predicted sublimons resulting from recombination across short repeats. Many mitochondrial gene products exhibit extraordinary levels of divergence in Viscum, indicative of highly relaxed if not positive selection. In addition, all Viscum mitochondrial protein genes have experienced a dramatic acceleration in synonymous substitution rates, consistent with the hypothesis of genomic streamlining in response to a high mutation rate but completely opposite to the pattern seen for the high-rate but enormous mitogenomes of Silene. In sum, the Viscum mitogenome possesses a unique constellation of extremely unusual features, a subset of which may be related to its parasitic lifestyle.

Comparative mitogenomics indicates respiratory competence in parasitic Viscum despite loss of complex I and extreme sequence divergence, and reveals horizontal gene transfer and remarkable variation in genome size.

BACKGROUND: Aerobically respiring eukaryotes usually contain four respiratory-chain complexes (complexes I-IV) and an ATP synthase (complex V). In several lineages of aerobic microbial eukaryotes, complex I has been lost, with an alternative, nuclear-encoded NADH dehydrogenase shown in certain cases to bypass complex I and oxidize NADH without proton translocation. The first loss of complex I in any multicellular eukaryote was recently reported in two studies; one sequenced the complete mitogenome of the hemiparasitic aerial mistletoe, Viscum scurruloideum, and the other sequenced the V. album mitogenome. The V. scurruloideum study reported no significant additional loss of mitochondrial genes or genetic function, but the V. album study postulated that mitochondrial genes encoding all ribosomal RNAs and proteins of all respiratory complexes are either absent or pseudogenes, thus raising questions as to whether the mitogenome and oxidative respiration are functional in this plant. RESULTS: To determine whether these opposing conclusions about the two Viscum mitogenomes reflect a greater degree of reductive/degenerative evolution in V. album or instead result from interpretative and analytical differences, we reannotated and reanalyzed the V. album mitogenome and compared it with the V. scurruloideum mitogenome. We find that the two genomes share a complete complement of mitochondrial rRNA genes and a typical complement of genes encoding respiratory complexes II-V. Most Viscum mitochondrial protein genes exhibit very high levels of divergence yet are evolving under purifying, albeit relaxed selection. We discover two cases of horizontal gene transfer in V. album and show that the two Viscum mitogenomes differ by 8.6-fold in size (66 kb in V. scurruloideum; 565 kb in V. album). CONCLUSIONS: Viscum mitogenomes are extraordinary compared to other plant mitogenomes in terms of their wide size range, high rates of synonymous substitutions, degree of relaxed selection, and unprecedented loss of respiratory complex I. However, contrary to the initial conclusions regarding V. album, both Viscum mitogenomes possess conventional sets of rRNA and, excepting complex I, respiratory genes. Both plants should therefore be able to carry out aerobic respiration. Moreover, with respect to size, the V. scurruloideum mitogenome has experienced a greater level of reductive evolution.

Reading between the vines: Hosts as islands for extreme holoparasitic plants.

PREMISE OF THE STUDY: Partitioning of population genetic variation in plants may be affected by numerous factors including life history and dispersal characteristics. In parasitic plants, interactions with host populations may be an additional factor influencing partitioning. To test for hierarchical population genetic patterns related to obligate endoparasitism, we studied three species of Rafflesiaceae, which grow as extremely reduced endophytes infecting Tetrastigma vines in Southeast Asia. METHODS: Microsatellite markers were developed and multilocus genotypes were determined for Rafflesia cantleyi, Rafflesia tuan-mudae, and Sapria himalayana and each of their Tetrastigma hosts. Relatedness among parasite individuals was estimated, and AMOVAs were used to determine levels of population genetic subdivision. KEY RESULTS: Microsatellite genotypes for 340 paired parasite and host samples revealed that host vines were infected by numerous Rafflesiaceae individuals that may spread for up to 14 m within stem tissues. Surprisingly, Rafflesiaceae parasites within a given host are significantly more closely related to each other than individuals of the same species in other host individuals. The pattern of hierarchical population genetic subdivision we detected across species is likely due to limited seed dispersal with reinfection of natal host vines. CONCLUSIONS: These findings demonstrate common population genetic patterns between animal and plant parasites, potentially indicating advantages of close relatives infecting hosts. This study also has important conservation implications for Rafflesiaceae since our data suggest that destruction of a single infected host vine could result in large genetic losses.

Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates.

In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (k(cat)/K(M)) for competing substrates, even though adaptive substitutions may affect K(M) and k(cat) separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities.

The Mutational Road not Taken: Using Ancestral Sequence Resurrection to Evaluate the Evolution of Plant Enzyme Substrate Preferences.

We investigated the flowering plant salicylic acid methyl transferase (SAMT) enzyme lineage to understand the evolution of substrate preference change. Previous studies indicated that a single amino acid replacement to the SAMT active site (H150M) was sufficient to change ancestral enzyme substrate preference from benzoic acid to the structurally similar substrate, salicylic acid (SA). Yet, subsequent studies have shown that the H150M function-changing replacement did not likely occur during the historical episode of enzymatic divergence studied. Therefore, we reinvestigated the origin of SA methylation preference here and additionally assessed the extent to which epistasis may act to limit mutational paths. We found that the SAMT lineage of enzymes acquired preference to methylate SA from an ancestor that preferred to methylate benzoic acid as previously reported. In contrast, we found that a different amino acid replacement, Y267Q, was sufficient to change substrate preference with others providing small positive-magnitude epistatic improvements. We show that the kinetic basis for the ancestral enzymatic change in substate preference by Y267Q appears to be due to both a reduced specificity constant, kcat/KM, for benzoic acid and an improvement in KM for SA. Therefore, this lineage of enzymes appears to have had multiple mutational paths available to achieve the same evolutionary divergence. While the reasons remain unclear for why one path was taken, and the other was not, the mutational distance between ancestral and descendant codons may be a factor.

Accelerated rates of floral evolution at the upper size limit for flowers.

Evolutionary theory explains phenotypic change as the result of natural selection, with constraint limiting the direction, magnitude, and rate of response [1]. Constraint is particularly likely to govern evolutionary change when a trait is at perceived upper or lower limits. Macroevolutionary rates of floral-size change are unknown for any angiosperm family, but it is predicted that rates should be diminished near the upper size limit of flowers, as has been shown for mammal body mass [2]. Our molecular results show that rates of floral-size evolution have been extremely rapid in the endoholoparasite Rafflesia, which contains the world's largest flowers [3]. These data provide the first estimates of macroevolutionary rates of floral-size change and indicate that in this lineage, floral diameter increased by an average of 20 cm (and up to 90 cm)/million years. In contrast to our expectations, it appears that the magnitude and rate of floral-size increase is greater for lineages with larger flowered ancestors. This study suggests that constraints on rates of floral-size evolution may not be limiting in Rafflesia, reinforcing results of artificial- and natural-selection studies in other plants that demonstrated the potential for rapid size changes [4-6].

Enzymatic, expression and structural divergences among carboxyl O-methyltransferases after gene duplication and speciation in Nicotiana.

Methyl salicylate and methyl benzoate have important roles in a variety of processes including pollinator attraction and plant defence. These compounds are synthesized by salicylic acid, benzoic acid and benzoic acid/salicylic acid carboxyl methyltransferases (SAMT, BAMT and BSMT) which are members of the SABATH gene family. Both SAMT and BSMT were isolated from Nicotiana suaveolens, Nicotiana alata, and Nicotiana sylvestris allowing us to discern levels of enzyme divergence resulting from gene duplication in addition to species divergence. Phylogenetic analyses showed that Nicotiana SAMTs and BSMTs evolved in separate clades and the latter can be differentiated into the BSMT1 and the newly established BSMT2 branch. Although SAMT and BSMT orthologs showed minimal change coincident with species divergences, substantial evolutionary change of enzyme activity and expression patterns occurred following gene duplication. After duplication, the BSMT enzymes evolved higher preference for benzoic acid (BA) than salicylic acid (SA) whereas SAMTs maintained ancestral enzymatic preference for SA over BA. Expression patterns are largely complementary in that BSMT transcripts primarily accumulate in flowers, leaves and stems whereas SAMT is expressed mostly in roots. A novel enzyme, nicotinic acid carboxyl methyltransferase (NAMT), which displays a high degree of activity with nicotinic acid was discovered to have evolved in N. gossei from an ancestral BSMT. Furthermore a SAM-dependent synthesis of methyl anthranilate via BSMT2 is reported and contrasts with alternative biosynthetic routes previously proposed. While BSMT in flowers is clearly involved in methyl benzoate synthesis to attract pollinators, its function in other organs and tissues remains obscure.

Mitochondrial DNA suggests at least 11 origins of parasitism in angiosperms and reveals genomic chimerism in parasitic plants.

BACKGROUND: Some of the most difficult phylogenetic questions in evolutionary biology involve identification of the free-living relatives of parasitic organisms, particularly those of parasitic flowering plants. Consequently, the number of origins of parasitism and the phylogenetic distribution of the heterotrophic lifestyle among angiosperm lineages is unclear. RESULTS: Here we report the results of a phylogenetic analysis of 102 species of seed plants designed to infer the position of all haustorial parasitic angiosperm lineages using three mitochondrial genes: atp1, coxI, and matR. Overall, the mtDNA phylogeny agrees with independent studies in terms of non-parasitic plant relationships and reveals at least 11 independent origins of parasitism in angiosperms, eight of which consist entirely of holoparasitic species that lack photosynthetic ability. From these results, it can be inferred that modern-day parasites have disproportionately evolved in certain lineages and that the endoparasitic habit has arisen by convergence in four clades. In addition, reduced taxon, single gene analyses revealed multiple horizontal transfers of atp1 from host to parasite lineage, suggesting that parasites may be important vectors of horizontal gene transfer in angiosperms. Furthermore, in Pilostyles we show evidence for a recent host-to-parasite atp1 transfer based on a chimeric gene sequence that indicates multiple historical xenologous gene acquisitions have occurred in this endoparasite. Finally, the phylogenetic relationships inferred for parasites indicate that the origins of parasitism in angiosperms are strongly correlated with horizontal acquisitions of the invasive coxI group I intron. CONCLUSION: Collectively, these results indicate that the parasitic lifestyle has arisen repeatedly in angiosperm evolutionary history and results in increasing parasite genomic chimerism over time.

Positive selection for single amino acid change promotes substrate discrimination of a plant volatile-producing enzyme.

We used a combined evolutionary and experimental approach to better understand enzyme functional divergence within the SABATH gene family of methyltransferases (MTs). These enzymes catalyze the formation of a variety of secondary metabolites in plants, many of which are volatiles that contribute to floral scent and plant defense such as methyl salicylate and methyl jasmonate. A phylogenetic analysis of functionally characterized members of this family showed that salicylic acid methyltransferase (SAMT) forms a monophyletic lineage of sequences found in several flowering plants. Most members of this lineage preferentially methylate salicylic acid (SA) as compared with the structurally similar substrate benzoic acid (BA). To investigate if positive selection promoted functional divergence of this lineage of enzymes, we performed a branch-sites test. This test showed statistically significant support (P<0.05) for positive selection in this lineage of MTs (dN/dS=10.8). A high posterior probability (pp=0.99) identified an active site methionine as the only site under positive selection in this lineage. To investigate the potential catalytic effect of this positively selected codon, site-directed mutagenesis was used to replace Met with the alternative amino acid (His) in a Datura wrightii floral-expressed SAMT sequence. Heterologous expression of wild-type and mutant D. wrightii SAMT in Escherichia coli showed that both enzymes could convert SA to methyl salicylate and BA to methyl benzoate. However, competitive feeding with equimolar amounts of SA and BA showed that the presence of Met in the active site of wild-type SAMT resulted in a >10-fold higher amount of methyl salicylate produced relative to methyl benzoate. The Met156His-mutant exhibited little differential preference for the 2 substrates because nearly equal amounts of methyl salicylate and methyl benzoate were produced. Evolution of the ability to discriminate between the 2 substrates by SAMT may be advantageous for efficient production of methyl salicylate, which is important for pollinator attraction as well as pathogen and herbivore defense. Because BA is a likely precursor for the biosynthesis of SA, SAMT might increase methyl salicylate levels directly by preferential methylation and indirectly by leaving more BA to be converted into SA.

Evidence for positive selection on the floral scent gene isoeugenol-O-methyltransferase.

Isoeugenol-O-methyltransferase (IEMT) is an enzyme involved in the production of the floral volatile compounds methyl eugenol and methyl isoeugenol in Clarkia breweri (Onagraceae). IEMT likely evolved by gene duplication from caffeic acid-O-methyltransferase followed by amino acid divergence, leading to the acquisition of its novel function. To investigate the selective context under which IEMT evolved, maximum likelihood methods that estimate variable d(N)/d(S) ratios among lineages, among sites, and among a combination of both lineages and sites were utilized. Statistically significant support was obtained for a hypothesis of positive selection driving the evolution of IEMT since its origin. Subsequent Bayesian analyses identified several sites in IEMT that have experienced positive selection. Most of these positions are in the active site of IEMT and have been shown by site-directed mutagenesis to have large effects on substrate specificity. Although the selective agent is unknown, the adaptive evolution of this gene may have resulted in increased effectiveness of pollinator attraction or herbivore repellence.

Mitochondrial DNA sequences reveal the photosynthetic relatives of Rafflesia, the world's largest flower.

All parasites are thought to have evolved from free-living ancestors. However, the ancestral conditions facilitating the shift to parasitism are unclear, particularly in plants because the phylogenetic position of many parasites is unknown. This is especially true for Rafflesia, an endophytic holoparasite that produces the largest flowers in the world and has defied confident phylogenetic placement since its discovery >180 years ago. Here we present results of a phylogenetic analysis of 95 species of seed plants designed to infer the position of Rafflesia in an evolutionary context using the mitochondrial gene matR (1,806 aligned base pairs). Overall, the estimated phylogenetic tree is highly congruent with independent analyses and provides a strongly supported placement of Rafflesia with the order Malpighiales, which includes poinsettias, violets, and passionflowers. Furthermore, the phylogenetic placement of Mitrastema, another enigmatic, holoparasitic angiosperm with the order Ericales (which includes blueberries and persimmons), was obtained with these data. Although traditionally classified together, Rafflesia and Mitrastema are only distantly related, implying that their endoparasitic habits result from convergent evolution. Our results indicate that the previous significant difficulties associated with phylogenetic placement of holoparasitic plants may be overcome by using mitochondrial DNA so that a broader understanding of the origins and evolution of parasitism may emerge.

Comparative genomic analyses of frog virus 3, type species of the genus Ranavirus (family Iridoviridae).

Frog virus 3 (FV3) is the type species member of the genus Ranavirus (family Iridoviridae). To better understand the molecular mechanisms involved in the replication of FV3, including transcription of its highly methylated DNA genome, we have determined the complete nucleotide sequence of the FV3 genome. The FV3 genome is 105903 bp long excluding the terminal redundancy. The G + C content of FV3 genome is 55% and it encodes 98 nonoverlapping potential open reading frames (ORFs) containing 50-1293 amino acids. Eighty-four ORFs have significant homology to known proteins of other iridoviruses, whereas twelve of these unique FV3 proteins do not share homology to any known protein. A microsatellite containing a stretch of 34 tandemly repeated CA dinucleotide in a noncoding region was detected. To date, no such sequence has been reported in any animal virus.