Capturing the geometry,function, and evolution of enzymes with 3D templates

Structural templates are 3D signatures representing protein functional sites, such as ligand binding cavities, metal coordination motifs, or catalytic sites. Here we explore methods to generate template libraries and algorithms to query structures for conserved 3D motifs. Applications of templates are discussed, as well as some exemplar cases for examining evolutionary links in enzymes. We also introduce the concept of using more than one template per structure to represent flexible sites, as an approach to better understand catalysis through snapshots captured in enzyme structures. Functional annotation from structure is an important topic that has recently resurfaced due to the new more accurate methods of protein structure prediction. Therefore, we anticipate that template‐based functional site detection will be a powerful tool in the task of characterizing a vast number of new protein models.     

Looking at enzymes from the inside out: the proximity of catalytic residues to the molecular centroid can be used for detection of active sites and enzyme-ligand interfaces

Analysis of the distances of the exposed residues in 175 enzymes from the centroids of the molecules indicates that catalytic residues are very often found among the 5% of residues closest to the enzyme centroid. This property of catalytic residues is implemented in a new prediction algorithm (named EnSite) for locating the active sites of enzymes and in a new scheme for re-ranking enzyme-ligand docking solutions. EnSite examines only 5% of the molecular surface (represented by surface dots) that is closest to the centroid, identifying continuous surface segments and ranking them by their area size. EnSite ranks the correct prediction 1-4 in 97% of the cases in a dataset of 65 monomeric enzymes (rank 1 for 89% of the cases) and in 86% of the cases in a dataset of 176 monomeric and multimeric enzymes from all six top-level enzyme classifications (rank 1 in 74% of the cases). Importantly, identification of buried or flat active sites is straightforward because EnSite "looks" at the molecular surface from the inside out. Detailed examination of the results indicates that the proximity of the catalytic residues to the centroid is a property of the functional unit, defined as the assembly of domains or chains that form the active site (in most cases the functional unit corresponds to a single whole polypeptide chain). Using the functional unit in the prediction further improves the results. The new property of active sites is also used for re-evaluating enzyme-inhibitor unbound docking results. Sorting the docking solutions by the distance of the interface to the centroid of the enzyme improves remarkably the ranks of nearly correct solutions compared to ranks based on geometric-electrostatic-hydrophobic complementarity scores.     

Understanding the substituent effect on the acidity of alcohols and para-substituted phenols

We carried out Hartree–Fock (HF) and density functional theory calculations on the conjugated bases of phenols and alcohols for 23 compounds and analysed their acid–base behaviour using molecular orbital (MO) energies and their dependence on solvent effects. Despite the well-known correlation between highest-occupied MO (HOMO) energies and proton affinity (PA), we observed that HOMO energies are inadequate to describe the acid–base behaviour of these compounds. Therefore, we established a criterion to identify the best frontier MO for describing PA values and also to understand why the HOMO approach fails. The MO that fits our criterion provided very good correlations with PA values, much better than those obtained by the HOMO energies. Since the frontier MOs are those which drive the acid–base reactions in each compound, they were called frontier effective-for-reaction MOs, or FERMOs. By using the FERMO concept, the reactions that are HOMO driven, and those that are not, can be better explained, independent of the calculation method used, since both HF and Kohn–Sham methodologies lead to the same FERMO.     

Prediction of catalytic residues in enzymes based on known tertiary structure,stability profile,and sequence conservation

The catalytic or functionally important residues of a protein are known to exist in evolutionarily constrained regions. However, the patterns of residue conservation alone are sometimes not very informative, depending on the homologous sequences available for a given query protein. Here, we present an integrated method to locate the catalytic residues in an enzyme from its sequence and structure. Mutations of functional residues usually decrease the activity, but concurrently often increase stability. Also, catalytic residues tend to occupy partially buried sites in holes or clefts on the molecular surface. After confirming these general tendencies by carrying out statistical analyses on 49 representative enzymes, these data together with amino acid conservation were evaluated. This novel method exhibited better sensitivity in the prediction accuracy than traditional methods that consider only the residue conservation. We applied it to some so-called "hypothetical" proteins, with known structures but undefined functions. The relationships among the catalytic, conserved, and destabilizing residues in enzymatic proteins are discussed.     

Biological roles of specific peptides in enzymes

It has recently been shown (Kunik et al., PLOS Comput Biol 2007;3(8):e167) that the occurrence of specific peptides (SPs) on sequences of enzymes allows for accurate EC classification of enzymes. We inquire whether these SPs play important roles in bringing about the enzymatic function. This is assessed by cross-checking the occurrence of SPs on enzymes with Swiss-Prot annotations and PDB spatial structures of enzymes. Analyzing the coverage of functional annotations of enzymes, we demonstrate that SPs contain major fractions of all annotated features. This result is statistically highly significant and associates over 10% of all SPs with important biological markers. Concentrating on DNA binding regions, relevant to LexA repressor enzymes, we find interesting coverage patterns. Moreover, for the same data, we demonstrate that SPs allow for subclassification of the relevant bacteria into phylogenetic classes. An analysis of mutagen annotations on SPs appearing on all enzymes leads to the conclusion that mutations on SPs tend to damage the enzymatic function much more than expected from a background model, hence SPs are of high importance to enzymatic functions. SPs that lie in 3D pockets that are shared by active and binding sites, are shown to be significantly enriched by glycine, leading to the hypothesis that they are responsible for conformational plasticity. Finally we show that SPs can partially resolve outstanding difficult problems of convergent evolution by representing correctly enzyme functions in spite of remote homologies in sequence and in structure.     

蛋白质酶功能分析和预测方法的进展和前瞻

蛋白质酶是生物体内最重要的生物分子之一。对酶的功能进行系统研究具有重要的科学研究价值和工业应用意义,近年来,以计算机技术为基础的酶功能预测的方法不断发展与完善。基于此背景,本文总结了基于计算方法的酶功能分析与预测的主要方法,包括酶结合位点、分子对接、动力学模拟以及分子设计等内容。同时,本文也对相应的发展趋势进行讨论和展望。     

On the structure and operation of type I DNA restriction enzymes.

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.     

Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12-GA9, and GA53-GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2beta-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3beta-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.     

Peroxide-utilizing biocatalysts: structural and functional diversity of heme-containing enzymes

Heme-containing enzymes, such as peroxidases, catalase and peroxygenase P450 all utilize peroxides for their specific reactions. A variety of reactions catalyzed by such heme-containing enzymes involve a common, highly reactive intermediate, the so-called compound I (oxo-ferryl porphyrin pi-cation radical), which is generated via the reaction of peroxide with a ferric heme iron. However, the main reaction catalyzed by the heme-containing enzyme is determined by the accessibility of substrates to their active sites. Using the accumulated knowledge, we delineate a view, in which machineries of the heme-containing enzymes, especially the heme distal side structures, precisely regulate their functions in terms of sharing a common reactive intermediate. We also show the possibility that a hemoprotein of one functionality can be engineered to that with another functionality by modifying the heme distal side elements, on the basis of molecular-based mechanistic and structural data on these peroxide-utilizing enzymes.     

Structure based prediction of functional sites with potential inhibitors to Nudix enzymes from disease causing microbes

The functional sites were predicted for Nudix enzymes from pathogenic microorganisms such as Streprococcus pneumonia (2B06) and Enterococcus faecalis (2AZW). Their structures are already determined, however, no data is reported about their functional sites, substrates and inhibitors. Therefore, we report prediction of functional sites in these Nudix enzymes via Geometric Invariant (GI) technique (Construct different geometries of peptides which remain unchanged). The GI method enumerated 2B06: RA57, EA58, EA61, EA62 and 2AZW: RA62, EA63, EA66, EA67 as putative functional sites in these Nudix enzymes. In addition, the substrate was predicted via Molecular docking (Docking of substrates against whole structure of Nudix enzymes). The substrate ADP-Ribose was docked with the Nudix enzymes, 2B06 (Docking energy -15.68 Kcal/mol) and 2AZW (Docking energy -10.86 Kcal/mol) with the higher affinity and the lower docking energy as compared to other substrates. The residues EA62 in 2B06 and RA62 in 2AZW make hydrogen bonds with the ADP-ribose. Furthermore, we screened 51 inhibitor compounds against structures of 2B06 and 2AZW. The inhibitor compounds AMPCPR and CID14258187 were docked well as compared to other compounds. The compound CID14258187 was also in agreement with Lipinski rule of 5 for drug likeness properties. Therefore, our findings of functional sites, substrates and inhibitors for these Nudix enzymes may help in structure based drug designing against Streprococcus pneumonia and Enterococcus faecalis.     

Structural enzymology of carbohydrate-active enzymes: implications for the post-genomic era

Simple and complex carbohydrates have been described as "the last frontier of molecular and cell biology". The enzymes that are required for the synthesis and degradation of these compounds provide an enormous challenge in the post-genomic era. This reflects both the extreme chemical and functional diversity of sugars and the difficulties in characterizing both the substrates and the enzymes themselves. The vast myriad of enzymes involved in the synthesis, modification and degradation of oligosaccharides and polysaccharides is only just being unveiled by genomic sequencing. These so-called "carbohydrate-active enzymes" lend themselves to classification by sensitive sequence similarity detection methods. The modularity, often extremely complex, of these enzymes must first be dissected and annotated before high throughput characterization or "structural genomics" approaches may be employed. Once achieved, modular analysis also permits collation of a detailed "census" of carbohydrate-active enzymes for a whole organism or throughout an ecosystem. At the structural level, improvements in X-ray crystallography have opened up a three-dimensional understanding of the way these enzymes work. The mechanisms of many of the glycoside hydrolase families are becoming clearer, yet glycosyltransferases are only slowly revealing their secrets. What is clear from the genomic and structural data is that if we are to harness the latent power of glycogenomics, scientists must consider distant sequence relatives revealed by the sequence families or other sensitive detection methods.     

Redox regulation of SUMO enzymes is required for ATM activity and survival in oxidative stress

To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox‐sensitive proteins. We previously identified SUMO (small ubiquitin‐related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox‐regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM–Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS‐producing chemotherapeutic drugs etoposide/Vp16 and Ara‐C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress.     

Structural and functional characterization of the human DNA repair helicase XPD by comparative molecular modeling and site-directed mutagenesis of the bacterial repair protein UvrB

A molecular model for the human nucleotide excision repair protein, XPD, was developed based on the structural and functional relationship of the protein with a bacterial nucleotide excision repair (NER) protein, UvrB. Whereas XPD does not share significant sequence identity with UvrB, the proteins share seven highly conserved helicase motifs that define a common protein structural template. They also have similar functional roles in their ATPase activity and the ability to unwind DNA and verify damaged strands in the process of NER. The validity of using the crystal structure of UvrB as a template for the development of an XPD model was tested by mimicking human disease-causing mutations (XPD: R112H, D234N, R601L) in UvrB (E110R, D338N, R506A) and by mutating two highly conserved residues (XPD, His-237 and Asp-609; UvrB, H341A and D510A). The XPD structural model can be employed in understanding the molecular mechanism of XPD human disease causing mutations. The value of this XPD model demonstrates the generalized approach for the prediction of the structure of a mammalian protein based on the crystal structure of a structurally and functionally related bacterial protein sharing extremely low sequence identity (<15%).     

Excited state intermolecular hydrogen bond’s effect on the luminescent behaviour of the 2D covalent organic framework (PPy-COF): A TDDFT insight

The theoretical investigation of electronically excited stated intermolecular hydrogen bonding dynamics of the 2D luminescent polypyrene covalent organic framework and methanol molecule (PPy-COF-MeOH) was performed using the density functional theory (DFT) and time-dependent (TD-DFT) method. The strengthening of Hydrogen bonds C-H---O-H and B-O---H-O upon photoexcitation was confirmed via comparison of geometric structures, electronic transition energies, ¹H-NMR, binding energies, UV-Vis and infrared spectra in S0 and S1 states. Frontier molecular orbitals (MOs) analysis, electronic configuration, Mulliken charge analysis; and the charge density variation in hydrogen bonding proximity demonstrated that the strengthened hydrogen bonds facilitate the nonradiative path which may consequently proceed the luminescence quenching. Hence, the molecular material property prediction package (MOMAP) programme verified the fluorescence quenching because PPy-COF-MeOH complex showed a lower fluorescent rate constant compared to isolated PPy-COF fragment. The S1-T1 energy gap analysis also revealed the possibility of the Intersystem crossing (ISC). Above results significantly highlighted the role of the hydrogen bonding dynamics on luminescence property of the PPy-COF.     

Structural and biochemical studies of the distinct activity profiles of Rai1 enzymes

Recent studies showed that Rai1 and its homologs are a crucial component of the mRNA 5′-end capping quality control mechanism. They can possess RNA 5′-end pyrophosphohydrolase (PPH), decapping, and 5′-3′ exonuclease (toward 5′ monophosphate RNA) activities, which help to degrade mRNAs with incomplete 5′-end capping. A single active site in the enzyme supports these apparently distinct activities. However, each Rai1 protein studied so far has a unique set of activities, and the molecular basis for these differences are not known. Here, we have characterized the highly diverse activity profiles of Rai1 homologs from a collection of fungal organisms and identified a new activity for these enzymes, 5′-end triphosphonucleotide hydrolase (TPH) instead of PPH activity. Crystal structures of two of these enzymes bound to RNA oligonucleotides reveal differences in the RNA binding modes. Structure-based mutations of these enzymes, changing residues that contact the RNA but are poorly conserved, have substantial effects on their activity, providing a framework to begin to understand the molecular basis for the different activity profiles.     

Identification of substrate binding sites in enzymes by computational solvent mapping

Enzyme structures determined in organic solvents show that most organic molecules cluster in the active site, delineating the binding pocket. We have developed algorithms to perform solvent mapping computationally, rather than experimentally, by placing molecular probes (small molecules or functional groups) on a protein surface, and finding the regions with the most favorable binding free energy. The method then finds the consensus site that binds the highest number of different probes. The probe-protein interactions at this site are compared to the intermolecular interactions seen in the known complexes of the enzyme with various ligands (substrate analogs, products, and inhibitors). We have mapped thermolysin, for which experimental mapping results are also available, and six further enzymes that have no experimental mapping data, but whose binding sites are well characterized. With the exception of haloalkane dehalogenase, which binds very small substrates in a narrow channel, the consensus site found by the mapping is always a major subsite of the substrate-binding site. Furthermore, the probes at this location form hydrogen bonds and non-bonded interactions with the same residues that interact with the specific ligands of the enzyme. Thus, once the structure of an enzyme is known, computational solvent mapping can provide detailed and reliable information on its substrate-binding site. Calculations on ligand-bound and apo structures of enzymes show that the mapping results are not very sensitive to moderate variations in the protein coordinates.