Evolutionary Analysis and Functional Characterization of Different Histidinol Phosphate Phosphatases

The elaborate metabolism of modern organisms raises the question of how such a complex system could have developed from a simple ancient prestage with a presumably very limited repertoire of enzymes. To answer this question various models have been put forward. A popular model assumes that the promiscuous side activities of enzymes represent a starting point for new reactions and reaction sequences from which novel enzymes can develop through gene duplication and divergent evolution. A remnant of these duplication events are the superfamilies of related enzymes, which have the same fold and may also have functional similarities but can often catalyze different reactions or catalyze the same type of reaction on different substrates. One of the most widespread superfamilies is the so-called haloacid dehalogenase (HAD) superfamily which mainly consists of phosphatases and phosphonatases that act on a wide variety of substrates. Substrate specificity is mainly achieved by highly variable cap modules which are inserted into a core Rossmann fold and cover the active site, both enabling specific interactions with the substrate and shielding the reaction center from bulk solvent. Due to the wide distribution of the HAD superfamily, it is assumed that it represents one of the oldest superfamilies. It is further assumed that various enzymes from the HAD superfamily that differed from each other by the aforementioned caps were already present in the last common ancestor of all cellular organisms (LUCA). A HAD enzyme with a comparatively original fold is the histidinol phosphate phosphatase (HolPase) from Escherichia coli (ecHisB-N). The HolPase is part of histidine biosynthesis, where it catalyzes the penultimate step, namely the dephosphorylation of histidinol phosphate to histidinol. Although this pathway is identical in all histidine-synthesizing species, which is why it is assumed that it was already present in LUCA, the HolPases of different species differ significantly and so far, HolPases from three different superfamilies have been identified. All the other enzymes from histidine biosynthesis are however conserved across different species, which means that the HolPases are probably evolutionary younger than the other enzymes of this pathway. This observation raises several questions, which are addressed in the present work. In the first part, the question regarding the evolutionary origin of ecHisB-N was raised. The fact that this enzyme is not conserved in histidine-synthesizing species, while belonging to a very old protein superfamily, indicates that ecHisB-N evolved from a more ancestral enzyme, possibly a primordial phosphatase. In previous work, it has been argued that ecHisB-N and its closest homologue D-glycero-D-manno-heptose-1,7-bisphosphat-7-phosphatase (GmhB) were derived from the same promiscuous phosphatase. GmhB variants catalyze the hydrolysis of the two anomers of D-glycero-D-manno-heptose-1,7-bisphosphat (αHBP or βHBP), with one anomer usually being highly preferred by αGmhB or βGmhB, respectively. We found that ecHisB-N shows promiscuous activity for βHBP but not for αHBP, while the βGmhB from Crassaminicella sp. showed a promiscuous activity for HolP. Consistent with this, in a combined phylogenetic tree of αGmhB, βGmhB, and HisB-N sequences, HisB-N sequences formed a compact subcluster derived from βGmhBs. To analyze the properties of the precursors, several enzymes were resurrected by ancestral sequence reconstruction, and their functionality was tested in vitro. In this analysis, a promiscuous HolPase activity could already be detected in the ancestral enzymes belonging to nodes that predate the functional divergence of βGmhB and HisB-N. This HolPase activity was significantly increased in enzymes that belong to younger nodes and from which only modern HisB-N enzymes are derived. This increase in the catalytic efficiency of HolP turnover is reflected in the shape and electrostatics of the active site as predicted by AlphaFold. Finally, a revised model for the evolution of HisB-N from an ancestral βGmhB was developed with the help of a detailed analysis of the phylogenetic tree. In agreement with the experimental data, a horizontal gene transfer of a promiscuous βGmhB enzyme from an ancestral δ-Proteobacterium to an ancestral γ-Proteobacterium is assumed. After the horizontal gene transfer, this βGmhB then most likely evolved into a modern HisB-N.In the second and third part, the question is asked whether there are other HolPases that are not directly related to HolPases which were reported so far and instead developed independently. Specifically, in the second part, a protein of the HAD superfamily from Pseudomonas aeruginosa (paHisN) is analyzed, for which a HolPase function was suggested on the basis of in vivo experiments that were reported in a recent publication. An analysis of the AlphaFold-predicted structure of paHisN in the present work confirmed its classification as a member of the HAD superfamily. A comparison to the HAD family HolPase ecHisB-N unveiled that the two structures differed significantly in their cap structures, indicating that paHisN was not derived from ecHisB-N or vice versa. Instead, paHisN showed considerable similarities to hosphoserine phosphatases (PSPases) both at the sequence level and regarding the protein fold, indicating a possible evolutionary relationship or overlapping functions. Subsequent characterization of the enzyme in vitro showed that the protein is present as a monomer in aqueous solution and has a melting temperature of 46°C. In addition, the assumed HolPase activity could be confirmed as the native function of this enzyme. Moreover, a promiscuous PSPase activity was discovered which supports the hypothesis of a distant relationship to this class of enzymes. To distinguish HolPases from PSPases among the homologues of paHisN, an alanine scan of the active site was performed to identify residues critical to HolPase function. The results of this alanine scan were used in combination with a sequence logo of PSPases to derive a fingerprint, consisting of a DxD motif and a tyrosine, which is assumed to be conclusive for a HolPase function. The subsequent analysis of a sequence similarity network (SSN) of homologous proteins led to the identification of numerous non-annotated proteins in β- and γ-Proteobacteria which contained this fingerprint. The conservation of this set of critical residues suggests, that these homologues are HolPases with similar properties as paHisN. This conclusion was cross-validated by a bioinformatic analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database which showed that for many β- and -Proteobacteria there was indeed no HolPase annotated. This finding supports the proposed annotation of the homologues as HolPases most likely identifying the last missing enzyme from histidine biosynthesis in these organisms. An additional bioinformatic analysis of all phyla revealed that there generally is a considerable knowledge-gap concerning the HolPase function as for 32 % of all histidine-synthesizing organisms the enzyme that catalyzes the HolPase reaction is not known. In archaea this knowledge-gap is especially pronounced as in this domain an annotated HolPase is missing in approximately two thirds of the histidine-synthesizing species. The third part is therefore dedicated to the search for the enzyme that catalyzes the HolPase reaction in the archaeal kingdom. In previous work, a gene between hisC and hisB that was annotated as a putative phosphatase from the HAD superfamily had been noticed in the archaeon Nitrosopumilus maritimus. The location of this gene within a cluster of genes from histidine biosynthesis and classification of its gene product as HAD protein made this a promising candidate for the missing HolPase. An analysis of sequence and the AlphaFold-predicted structure in the course of this work confirmed that this protein belongs to the HAD superfamily. However, the cap of this protein showed no similarity to the caps of either paHisN or ecHisB-N, indicating that all three proteins evolved independently. The comparison to a third HolPase from the HAD superfamily which was recently discovered in the archaeon Thermococcus onnurineus uncovered a limited sequence identity of 23.9% and a moderate similarity in the protein fold. While this indicated a distant relationship of the two proteins, the similarities were too low to infer a HolPase function for the protein from N. maritimus. Therefore, an in vitro characterization of the uncharacterized protein from N. maritimus was conducted which confirmed the suspected HolPase activity and showed that the protein exists as a monomer in aqueous solution and has a melting temperature of 37°C. In accordance to previously used nomenclature, nmHisN is therefore proposed as name for the protein from N. maritimus. The observed HolPase function of nmHisN, in combination with the distant relationship to the HolPase from T. onnurineus, suggested that these two proteins might be representative for a new class of significantly diverged HolPases which is widely distributed within the archaeal kingdom. To test this hypothesis, a fingerprint of HolPase defining residues was established on the basis of an alanine scan of the active site of nmHisN, which should allow for the identification of other archaeal HolPases. This fingerprint consisting of a DY motif, a lysine and a glutamate, which are assumed to be conclusive for a HolPase function of homologues of nmHisN. Afterwards, an SSN was created in which homologues of nmHisN were found in a wide variety of archaeal phyla. The homologues of two phyla, namely the Thaumarchaeota and the candidate phylum of Bathyarchaeota, contained a highly conserved fingerprint, strongly indicating that these proteins are HolPases. Moreover, homologues which contained three of the four fingerprint residues were identified in Euryarchaeota and, interestingly, also in bacterial δ-Proteobacteria. This indicates that these proteins also possess HolPase function. Taken together, the results of this part show that the HolPases from N. maritimus and T. onnurineus constitute a third type of HolPase from the HAD superfamily besides paHisN and ecHisB-N. This type of HolPase seems to be widely distributed among archaea which indicates its ancient history. The proposed annotation of these proteins as HolPases also helps to close this knowledge gap on the histidine biosynthesis of archaea. The fourth part of the work deals with functional transitions and the general evolvability of the HAD superfamily. Specifically, a possible functional transition from a PSPase to a HolPase was analyzed, as suggested in the case of the evolution of paHisN. For this, the E. coli PSPase ecSerB served as a model enzyme, which showed no measurable HolPase function in vitro. Several active site amino acids were randomized simultaneously and then tested for their ability to mediate growth of a ΔholPase strain on selective medium lacking histidine. After one round of random mutagenesis and selection, two enzyme variants were identified that exhibited continuously measurable HolPase activity in vitro with a catalytic efficiency of 1.7 M-1s-1 and 7.8 M-1s-1, respectively. In addition, the general evolvability of HAD enzymes was investigated using the promiscuous PSPase activity of ecHisB-N as case study. In previous work, directed evolution improved the promiscuous PSPase activity by about an order of magnitude. In this work, the activity could be increased by another order of magnitude by further random mutagenesis and selection. Here, D58 was identified as a key residue where mutation to asparagine alone resulted in a 60-fold improvement in PSPase activity. Both lines of experiment underscore the functional versatility of the HAD superfamily which can be easily evolved to new substrates through adaptations in the variable caps, while the basic catalytic machinery is preserved. Finally, based on the experimental data, a three-step model for the evolution of HolPases was proposed. It is assumed that HolP is hydrolyzed at a slow rate spontaneously in absence of an enzyme, mediated e.g., by Mg2+. It is furthermore assumed, that during the assembly of the histidine biosynthetic pathway the HolPase function was under the lowest selection pressure. The lack of conservation within the HolPases furthermore indicates that LUCA did not yet have a specialized HolPase. In the second step of evolution, with increasing genome size, the existence of one or more phosphatases with promiscuous HolPase activity became likely. However, specialized HolPases were most probably only established in a third step, after the three different kingdoms of life had diverged. After that, several horizontal gene transfers probably occurred, which is why homologous HolPases are now found in very distantly related organisms

The Effect of Non-lexical Verbal Signals on the Perceived Authenticity, Empathy and Understanding of a Listener

Active listening plays an important role in the relationship between clients and therapists. Here, we investigated whether variations of the confirmatory nonlexical verbal communication signal "mmh" influenced perceived authenticity, empathy and understanding of a listener. Eighty-one participants were in a conversation with an interviewer and reported about a difficult work experience. They were randomly assigned to one of three groups: The control group did not receive any verbal feedback from the interviewer; In one experimental group (1x-mmh), the interviewer uttered several monosyllabic confirmatory nonlexical verbal signals ("mmh") during the presentation; In a second experimental group (3x-mmh) the interviewer voiced several three syllable "mmh-mmh-mmh" while listening. All participants were then asked to rate the perceived authenticity, empathy and understanding of the interviewer. Participants in the 3x-mmh condition rated the interviewer to be significantly less authentic than those in the other two groups. No differences in reported empathy and understanding were found. The use of consecutive confirmatory nonlexical verbal signals ("mmh") - at least as currently implemented - may influence the perceived authenticity of a listener

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d-glycero-d-manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d-glycero-d-manno-heptose-1,7-bisphosphate (αHBP or βHBP) with a strong preference for one anomer (αGmhB or βGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for βHBP but not αHBP, while βGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, βGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from βGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous βGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N

Towards Photochromic Azobenzene‐Based Inhibitors for Tryptophan Synthase

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene‐based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto‐unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of similar to 100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures

In Silico Identification and Experimental Validation of Distal Activity-Enhancing Mutations in Tryptophan Synthase

Allostery is a central mechanism for the regulation of multi-enzyme complexes. The mechanistic basis that drives allosteric regulation is poorly understood but harbors key information for enzyme engineering. In the present study, we focus on the tryptophan synthase complex that is composed of TrpA and TrpB subunits, which allosterically activate each other. Specifically, we develop a rational approach for identifying key amino acid residues of TrpB distal from the active site. Those residues are predicted to be crucial for shifting the inefficient conformational ensemble of the isolated TrpB to a productive ensemble through intra-subunit allosteric effects. The experimental validation of the conformationally driven TrpB design demonstrates its superior stand-alone activity in the absence of TrpA, comparable to those enhancements obtained after multiple rounds of experimental laboratory evolution. Our work evidences that the current challenge of distal active site prediction for enhanced function in computational enzyme design has become within reach