Evolution Of Substrate Specificity Research Articles

Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His216-Gln219-Tyr221 motif in cellulose- and chitin-oxidizing MaAA10B toward Arg-Glu-Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H2O2, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H2O2. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

Abstract 1398 A very cool and novel phosphofructokinase sheds light on the evolution of substrate specificity and on the metabolic networks in our closest prokaryotic relatives

Abstract 1398 A very cool and novel phosphofructokinase sheds light on the evolution of substrate specificity and on the metabolic networks in our closest prokaryotic relatives

Evolution of substrate specificity in a maize anthranilate methyltransferase

Secondary metabolites play a key role in mediating pollination and plant defense against herbivory. One such metabolite, methyl anthranilate, is a volatile compound responsible for the recognizable scent of Concord grapes. Methyl anthranilate is commercially used as a bird repellent for golf courses and as a flavoring agent for grape‐flavored foods, beverages, and pharmaceuticals. A variety of plant species such as Zea mays (maize) and Vitis labrusca (fox grape) synthesize methyl anthranilate from anthranilate, an intermediate in tryptophan biosynthesis. Methyl anthranilate biosynthesis is a classic example of convergent evolution; in maize, this reaction is catalyzed by an anthranilate methyltransferase (AAMT1), while in grape, there is a two‐step pathway. Previous studies have found that the ZmAAMT1 SABATH methyltransferase likely evolved from methyltransferases that recognize the plant pathogen response hormone salicylic acid as a substrate. Using site‐directed mutagenesis, we introduced 12 independent missense mutations at 7 key active site residues with the intention of restoring substrate specificity for salicylic acid. This work will allow us to better trace and understand the evolution of anthranilate‐derived specialized metabolism in plants.

Evolution of substrate specificity and fungal symbiosis in filmy ferns (Hymenophyllaceae): a Bayesian approach for ambiguous character state reconstruction

Ferns, as landplants in general, originally form a symbiosis involving Arbuscular Mycorrhizal Fungi (AMF), which are prevalent in habitats directly connected to the soil, including low epiphytic sites, but are largely absent in the high epiphytic habitat. High probabilities of AMF should be expected in chiefly terrestrial lineages whereas truly, fully adapted epiphytic lineages should be lacking fungal endophytes or may have switched to other types of fungi; e.g. Dark Septate Endophytes (DSE), a morphological class of mostly unspecified fungi that is often found in epiphytic ferns and may include potentially symbiotic ascomycetes. We used a Bayesian approach for a comparison of the ambiguous character of preferred substrate with the incompletely known mycorrhization status for an ancient lineage of ferns, the Hymenophyllaceae or filmy ferns. The majority of the analysed 167 species prefers either the saxicolous, terrestrial or epiphytic habitat (differentiated into low and high epiphytic), but there are also many generalists without clear preference. For the whole family Hymenophyllaceae and one of the two main clades of the subfamily Trichomanoidae, the terrestrial habitat and AMF received the highest probability for representing the ancestral state. For the subfamily Hymenophylloidae, the low epiphytic habitat and DSE received the highest probability as ancestral state, whereas that for AMF was very low. The other main clade of the subfamily Trichomanoidae as well as the whole subfamily was found most likely to be originally terrestrial; but in both cases the probability values did not differ much from the values for low epiphytism, with all values around 50 ± 5%. The high epiphytic habitat, which in its present condition is thought to be possible only in angiosperm-dominated vegetation, did not receive high probability to be the ancestral state in any clade. This decouples the evolution of epiphytism in filmy ferns in time from the advent of the angiosperms, which is hypothesized to have triggered the radiation of all other major epiphytic fern lineages.

Evolution of substrate specificity in the Nucleobase-Ascorbate Transporter (NAT) protein family.

L-ascorbic acid (vitamin C) is an essential metabolite in animals and plants due to its role as an enzyme co-factor and antioxidant activity. In most eukaryotic organisms, L-ascorbate is biosynthesized enzymatically, but in several major groups, including the primate suborder Haplorhini, this ability is lost due to gene truncations in the gene coding for L-gulonolactone oxidase. Specific ascorbate transporters (SVCTs) have been characterized only in mammals and shown to be essential for life. These belong to an extensively studied transporter family, called Nucleobase-Ascorbate Transporters (NAT). The prototypic member of this family, and one of the most extensively studied eukaryotic transporters, is UapA, a uric acid-xanthine/H+ symporter in the fungus Aspergillus nidulans. Here, we investigate molecular aspects of NAT substrate specificity and address the evolution of ascorbate transporters apparently from ancestral nucleobase transporters. We present a phylogenetic analysis, identifying a distinct NAT clade that includes all known L-ascorbate transporters. This clade includes homologues only from vertebrates, and has no members in non-vertebrate or microbial eukaryotes, plants or prokaryotes. Additionally, we identify within the substrate-binding site of NATs a differentially conserved motif, which we propose is critical for nucleobase versus ascorbate recognition. This conclusion is supported by the amino acid composition of this motif in distinct phylogenetic clades and mutational analysis in the UapA transporter. Together with evidence obtained herein that UapA can recognize with extremely low affinity L-ascorbate, our results support that ascorbate-specific NATs evolved by optimization of a sub-function of ancestral nucleobase transporters.

The evolution of function within the Nudix homology clan.

ABSTRACTThe Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch‐specific adenine glycosylases (A/G‐specific adenine glycosylases), and nonenzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally‐based annotations. We manually constructed a structure‐guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 “select” Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy. Proteins 2017; 85:775–811. © 2016 Wiley Periodicals, Inc.

The Evolution of Substrate Specificity by tRNA Modification Enzymes.

All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. Over 100 different modifications have been described and every position in the purine and pyrimidine bases can be modified; often the sugar is also modified [1]. In tRNA, the function of modifications varies; some modulate global and/or local RNA structure, and others directly impact decoding and may be essential for viability. Whichever the case, the overall importance of modifications is highlighted by both their evolutionary conservation and the fact that organisms use a substantial portion of their genomes to encode modification enzymes, far exceeding what is needed for the de novo synthesis of the canonical nucleotides themselves [2]. Although some modifications occur at exactly the same nucleotide position in tRNAs from the three domains of life, many can be found at various positions in a particular tRNA and their location may vary between and within different tRNAs. With this wild array of chemical diversity and substrate specificities, one of the big challenges in the tRNA modification field has been to better understand at a molecular level the modes of substrate recognition by the different modification enzymes; in this realm RNA binding rests at the heart of the problem. This chapter will focus on several examples of modification enzymes where their mode of RNA binding is well understood; from these, we will try to draw general conclusions and highlight growing themes that may be applicable to the RNA modification field at large.

The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity.

ATP-binding cassette (ABC) transporters, although being ubiquitous in biology, often feature a subunit that is limited primarily to bacteria and archaea. This subunit, the substrate-binding protein (SBP), is a key determinant of the substrate specificity and high affinity of ABC uptake systems in these organisms. Most prokaryotes have many SBP-dependent ABC transporters that recognize a broad range of ligands from metal ions to amino acids, sugars and peptides. Herein, we review the structure and function of a number of more unusual SBPs, including an ABC transporter involved in the transport of rare furanose forms of sugars and an SBP that has evolved to specifically recognize the bacterial cell wall-derived murein tripeptide (Mtp). Both these examples illustrate that subtle changes in binding-site architecture, including changes in side chains not directly involved in ligand co-ordination, can result in significant alteration of substrate range in novel and unpredictable ways.

Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases.

BackgroundUnderstanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families.ResultsComparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site.ConclusionsThe structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-6834-7-109) contains supplementary material, which is available to authorized users.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Evolution of Substrate Specificity in a Recipient’s Enzyme Following Horizontal Gene Transfer

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism.

Ca2+ Induces Spontaneous Dephosphorylation of a Novel P5A-type ATPase

P5 ATPases constitute the least studied group of P-type ATPases, an essential family of ion pumps in all kingdoms of life. Although P5 ATPases are present in every eukaryotic genome analyzed so far, they have remained orphan pumps, and their biochemical function is obscure. We show that a P5A ATPase from barley, HvP5A1, locates to the endoplasmic reticulum and is able to rescue knock-out mutants of P5A genes in both Arabidopsis thaliana and Saccharomyces cerevisiae. HvP5A1 spontaneously forms a phosphorylated reaction cycle intermediate at the catalytic residue Asp-488, whereas, among all plant nutrients tested, only Ca(2+) triggers dephosphorylation. Remarkably, Ca(2+)-induced dephosphorylation occurs at high apparent [Ca(2+)] (K(i) = 0.25 mM) and is independent of the phosphatase motif of the pump and the putative binding site for transported ligands located in M4. Taken together, our results rule out that Ca(2+) is a transported substrate but indicate the presence of a cytosolic low affinity Ca(2+)-binding site, which is conserved among P-type pumps and could be involved in pump regulation. Our work constitutes the first characterization of a P5 ATPase phosphoenzyme and points to Ca(2+) as a modifier of its function.

Characterization of a hypotaurocyamine kinase from the protozoan, Phytophthora sojae, and its implications on the evolution of substrate specificity in the phosphagen kinase family

Phosphagen kinases (PK) are a family of enzymes that catalyze the reversible transfer of a high‐energy gamma phosphoryl group from ATP to a guanidino acceptor molecule. A novel hypotaurocyamine kinase (HTK) from the protozoan oomycete Phytophthora sojae (PsPK) was studied to determine substrate specificity, oligomerization state, and cooperativity. Isothermal titration calorimetry was used to determine the kinetic constants of PsPK reacting with taurocyamine and, less strongly, glycocyamine by titrating the guanidino substrate at varying concentrations into a solution containing the HTK and MgATP. The Km for taurocyamine and MgATP was found to be 0.59 mM and 3.78 mM, respectively, with a kcat of 34.66 s−1, while the Km for glycocyamine and MgATP was found to be 5.1 mM and 5.1 mM, respectively, with a kcat of 3.2 s−1. Size exclusion chromatography indicates PsPK is an oligomer and isothermal titration calorimetry suggests no cooperativity among subunits. Our results coupled with findings in Monosiga suggest oligomerization evolved first in the protozoa and that cooperativity sometime later. The data also indicate there is diverse substrate specificity in protozoan PKs previously thought to exist only in annelida. This project was made possible by the support of USDA grant (USDA 2011‐68004‐30104) and the Howard Hughes Medical Institute Undergraduate Science Education Program.

Iron-sulfur cluster engineering provides insight into the evolution of substrate specificity among sulfonucleotide reductases.

Assimilatory sulfate reduction supplies prototrophic organisms with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation via an ATP-dependent activation to form adenosine-5'-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes have no human homologue, rendering them attractive targets for the development of novel antibacterial drugs. APR and PAPR share sequence and structure homology as well as a common catalytic mechanism, but the enzymes are distinguished by two features, namely, the amino acid sequence of the phosphate-binding loop (P-loop) and an iron-sulfur cofactor in APRs. On the basis of the crystal structures of APR and PAPR, two P-loop residues are proposed to determine substrate specificity; however, this hypothesis has not been tested. In contrast to this prevailing view, we report here that the P-loop motif has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic, and kinetic analyses, we demonstrate that the iron-sulfur cluster cofactor enhances APS reduction by nearly 1000-fold, thereby playing a pivotal role in substrate specificity and catalysis. These findings offer new insights into the evolution of this enzyme family and extend the known functions of protein-bound iron-sulfur clusters.

High-throughput FACS method for directed evolution of substrate specificity.

High-throughput FACS method for directed evolution of substrate specificity.

Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates

Background: Understanding the networks of selective proteolysis that regulate complex biological systems requires an appreciation of the molecular mechanisms used to maintain substrate specificity. Human plasmin, a serine protease that promotes the dissolution of blood clots and is essential in maintaining normal hemostasis, is usually described as having broad substrate specificity. Recent evidence that plasmin also plays a key role in a variety of other important biological and pathological processes, however, has suggested that this description might need to be re-evaluated. Results: We used substrate phage display to elucidate optimal subsite occupancy for substrates of plasmin. We identified a peptide substrate that is cleaved 710,000-fold more efficiently by plasmin than a peptide containing the activation sequence of plasminogen. Plasmin achieves this unexpected, large differential activity even though both target sequences possess an arginine residue in the P1 position. We also demonstrate that proteolysis by plasmin can be targeted to an engineered protein substrate and that introduction of substrate sequences identified by phage display into plasminogen increases plasmin-mediated cleavage of the mutant 2000-fold. Conclusions: The specificity of plasmin is more tightly controlled than previously recognized; interactions with substrates at all subsites between S4 and S2′ contribute to catalysis. Furthermore, in contrast to most enzymes that exhibit positive selectivity for substrate, the evolution of substrate specificity by plasmin has apparently been dominated by a strong negative selection against development of autoactivation activity. This ‘negative selectivity’ avoids short-circuiting regulation of the fibrinolytic system and other important biological processes, and might be an important general mechanism for controlling protease cascades.

Phylogenetic Relationships of the Silver Saxifrages (Saxifraga, Sect. Ligulatae Haworth): Implications for the Evolution of Substrate Specificity, Life Histories, and Biogeography

The silver saxifrages (Saxifraga sect. Ligulatae Haworth; Saxifragaceae) exhibit remarkable variation of substrate specialization, with strictly calcicole to calcifuge species, as well as life histories which range from semelparity to iteroparity. They occur almost exclusively in the European mountain ranges and display high levels of endemism. Sequences from chloroplast and nuclear ribosomal DNA were obtained to resolve phylogenetic relationships among the silver saxifrages and related taxa and to gain insight into the evolution of substrate specificity, life history, and biogeography. The resulting phylogenies suggested that (1) Saxifraga sect. Ligulatae, as traditionally defined, does not constitute a monophyletic group; (2) lime-secreting hydathodes in calcifuge species apparently represent a secondary nonaptation; (3) semelparity evolved independently two or three times in the silver saxifrages and allied sections, possibly in response to climatic changes that occured during the Pleistocene; and (4) narrow endemics, for example S. cochlearis, likely evolved from the fragmentation of the widespread S. paniculata into refugial populations that became isolated during the glacial maxima of the Pleistocene.

Evolution of Substrate Specificities in the P-Type ATPase Superfamily

P-type ATPases make up a large superfamily of ATP-driven pumps involved in the transmembrane transport of charged substrates. We have performed an analysis of conserved core sequences in 159 P-type ATPases. The various ATPases group together in five major branches according to substrate specificity, and not according to the evolutionary relationship of the parental species, indicating that invention of new substrate specificities is accompanied by abrupt changes in the rate of sequence evolution. A hitherto-unrecognized family of P-type ATPases has been identified that is expected to be represented in all the major phyla of eukarya.

The glutathione S-transferase D genes. A divergently organized, intronless gene family in Drosophila melanogaster

We have characterized a cluster of glutathione S-transferase genes located at 87B on the Drosophila polytene chromosome near the heat shock genes, hsp70. These genes, designated gst Ds in the glutathione S-transferase gene superfamily, are closely linked within a approximately 60-kilobase DNA segment. The gene family has a minimum of eight intronless genes organized in divergent orientations. Two of the genes are probably GST pseudogenes in that their open reading frames are shorter than functional GSTs, and no RNAs from them have been detected thus far. The amino acid sequence identity among the functional genes ranges from 53 to 75% in pairwise comparisons. The intergenic regions are much more AT rich (63-73%) than the coding regions (41-52%), consistent with being promoter/regulatory sequences in Drosophila melanogaster. The mRNA size for each gene suggests that these genes are probably expressed individually from separate promoters. This is the first documentation of definitive physical linkage of a functional glutathione S-transferase multigene family. The genes are divergently organized, and a gradation of sequence similarity exists among the encoded GST isozymes. The patterns of sequence similarity in pairwise comparisons of the family members suggest that gene conversion may have played a role in the evolution of this GST multigene family. We propose that the Drosophila gst D genes provide a unique system for studying GST gene regulation, in vivo physiological functions, and evolution of substrate specificities with a global perspective. The gst D genes in other organisms should be intronless and can be isolated directly from genomic DNAs for functional analyses at the gene and protein levels.

Serological and genetical studies on the evolution of substrate specificity of flavone glycosyltransferase genes inSilene

The variation in flavone glycosylation patterns inSilene is the result of the expression of six genetic loci, which control either the presence of allozymes differing in substrate specificity or isozymes regulated differently during development. Serological studies showed that at least three of these six loci are evolutionarily related. The genetic mechanisms leading to these complicated variation patterns and the role of this polymorphism for the plant in its interaction with the environment are discussed.