Rotamer strain as a determinant of protein structural specificity

We present direct evidence for a change in protein structural specificity due to hydrophobic core packing. High resolution structural analysis of a designed core variant of ubiquitin reveals that the protein is in slow exchange between two conformations. Examination of side-chain rotamers indicates that this dynamic response and the lower stability of the protein are coupled to greater strain and mobility in the core. The results suggest that manipulating the level of side-chain strain may be one way of fine tuning the stability and specificity of proteins.     

Bioinformatic tools uncover the C-terminal strand of Rubisco's large subunit as hot-spot for specificity-enhancing mutations

Rubisco assumes the double role of accumulating biomass by fixing carbon dioxide to ribulose-1,5-bisphosphate and binding of molecular oxygen to the same substrate. The specificity factor of this mutually competitive activity, defined as the ratio of carboxylation to oxygenation efficiency, varies considerably for reasons which remain obscure. The explanation and the enhancement of specificity are of high theoretical and practical interest. Despite a wealth of structures and experimental findings, the systematic analysis of available data is still at its beginning. Here, we (a) present an analysis of sequences of the large subunit which reliably finds specificity-enhancing mutations and ranks them according to the probability of success. For mutations near the C-terminus, we (b) show by simulations that the positive influence they have on specificity can be explained by the time-window hypothesis.     

Measurements of the CO2/O2 specificity of ribulose 1, 5-bisphosphate carboxylase/oxygenase by 31P- and 1H-NMR

High resolution NMR spectroscopy has been demonstrated to be capable of measuring the CO2/O2 specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase. 31P-NMR provides a simple method for quantitatively determining the ratio of the products, 3-phosphoglycerate and phosphoglycolate. Both the specificity factor and degree of the reaction can be measured during the reaction without the need for complete consumption of ribulose bisphosphate or removal of it from the reaction mixture. Inorganic phosphate can also be detected and this may be used for monitoring the phosphatase activity and pH changes. Measurement by highly sensitive 1H-NMR is most time-efficient and is particularly suitable for the Rubisco with a high specificity factor. By optimizing the experimental conditions, it is possible to follow the simultaneous reactions in situ. The NMR method has been applied to three Rubisco enzymes with different values of specificity factor. Both 31P- and 1H-NMR gave similar results, agreeing with those previously reported by other methods.     

Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity

Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes—the acceleration of DNA cleavage and the expansion of sequence specificity—are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca²⁺ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme–DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein–DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.     

Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts

The Rubisco activase amino acid sequences of spinach and tobacco are 79% identical, yet the tobacco protein does not facilitate the activation of the uncarbamylated, ribulose bisphosphate bound form of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and vice versa. In contrast, combinations of the spinach Rubisco activase with Rubisco from non-Solanaceae species and combinations of tobacco Rubisco activase with Rubisco from other Solanaceae species are almost as effective as the analogous combination. To examine the basis of the preference of an activase protein for either Solanaceae or non-Solanaceae Rubisco, several recombinant chimeric proteins were obtained by combining regions from the cDNAs of spinach and tobacco activase and expression in Escherichia coli. The chimeric proteins were analyzed for ATP hydrolysis and ability to activate spinach and tobacco Rubisco. Comparisons of Rubisco preference with composition of the various activase chimeras indicate that the major determinants of Rubisco preference seem to be localized in the carboxyl-terminal region.     

An improved equation and assay for determining the CO2/O2 specificity for Rubisco

The determination of the CO₂/O₂ specificity factor (Ω) is very important to investigate the Rubisco carbon assimilation efficiency. In this paper, it is proved that previous formulae can introduce notable errors into calculating the CO₂/O₂ specificity factor (Ω) because CO₂ and O₂ are both the substrates and the mutually competitive inhibitors for Rubisco. A simple integrated equation is proposed to calculate the CO₂/O₂ specificity factor (Ω). On the other hand, previous multi-step procedures, including the manipulation of radioisotope (¹⁴C and/or ³H) and the chromatographic separation of the products, are inconvenient and may cause much random error. An improved assay procedure is presented therefore, which includes the spectrophotometric measurement of 3-phosphoglycerate-dependent NADH oxidation with a coupled enzyme system. This revised version was published online in June 2006 with corrections to the Cover Date.     

How various factors influence the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase

Temperature, activating metal ions, and amino-acid substitutions are known to influence the CO₂/O₂ specificity of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. However, an understanding of the physical basis for enzyme specificity has been elusive. We have shown that the temperature dependence of CO₂/O₂ specificity can be attributed to a difference between the free energies of activation for the carboxylation and oxygenation partial reactions. The reaction between the 2,3-enediolate of ribulose 1,5-bisphosphate and O₂ has a higher free energy of activation than the corresponding reaction of this substrate with CO₂. Thus, oxygenation is more responsive to temperature than carboxylation. We have proposed possible transition-state structures for the carboxylation and oxygenation partial reactions based upon the chemical natures of these two reactions within the active site. Electrostatic forces that stabilize the transition state of the carboxylation reaction will also inevitably stabilize the transition state of the oxygenation reaction, indicating that oxygenase activity may be unavoidable. Furthermore, the reduction in CO₂/O₂ specificity that is observed when activator Mg²⁺ is replaced by Mn²⁺ may be due to Mg²⁺ being more effective in neutralizing the negative charge of the carboxylation transition state, whereas Mn²⁺ is a transition-metal ion that can overcome the triplet character of O₂ to promote the oxygenation reaction.Abbreviations CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K_c K_mfor CO₂ - K_o K_mfor O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V_c V _max for carboxylation - V_o V _max for oxygenation     

Structural determinants and distribution of phosphate specificity in ribonucleotide reductases

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.     

Transgenic approaches to manipulate the environmental responses of the C3 carbon fixation cycle

The limitation to photosynthetic CO2 assimilation in C3 plants in hot, dry environments is dominated by ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) because CO2 availability is restricted and photorespiration is stimulated. Using a combination of genetic engineering and transgenic technology, three approaches to reduce photorespiration have been taken; two of these focused on increasing the carboxylation efficiency of Rubisco either by reducing the oxygenase reaction directly or by manipulating the Rubisco enzyme by concentrating CO2 in the region of Rubisco through the introduction of enzymes of the C4 pathway. The third approach attempted to reduce photorespiration directly by manipulation of enzymes in this pathway. The progress in each of these areas is discussed, and the most promising approaches are highlighted. Under saturating CO2 conditions, Rubisco did not limit photosynthesis, and limitation shifted to ribulose bisphosphate (RuBP) regeneration capacity of the C3 cycle. Transgenic analysis was used to identify the specific enzymes that may be targets for improving carbon fixation, and the way this may be exploited in the high CO2 future is considered.     

从酶的专一性看植酸酶与酸性磷酸酶的关系

迄今为止的资料表明,大多数植酸酶与酸性磷酸酶的关系密切。通过测定Km,即可判断只有以植酸（盐）为最适底物的酶（包括酸性磷酸酶）才是严格意义上的植酸酶。     

Substrate specificity of polyphenol oxidase

Abstract

The ubiquitous type-3 copper enzyme polyphenol oxidase (PPO) has found itself the subject of profound inhibitor research due to its role in fruit and vegetable browning and mammalian pigmentation. The enzyme itself has also been applied in the fields of bioremediation, biocatalysis and biosensing. However, the nature of PPO substrate specificity has remained elusive despite years of study. Numerous theories have been proposed to account for the difference in tyrosinase and catechol oxidase activity. The “blocker residue” theory suggests that bulky residues near the active site cover CuA, preventing monophenol coordination. The “second shell” theory suggests that residues distant (～8?Å) from the active site, guide and position substrates within the active site based on their properties e.g., hydrophobic, electrostatic. It is also hypothesized that binding specificity is related to oxidation mechanisms of the catalytic cycle, conferred by coordination of a conserved water molecule by other conserved residues. In this review, we highlight recent developments in the structural and mechanistic studies of PPOs and consolidate key concepts in our understanding toward the substrate specificity of PPOs.     

Rubisco specificity factor tends to be larger in plant species from drier habitats and in species with persistent leaves

The specificity factor of Rubisco is a measure of the relative capacities of the enzyme to catalyse carboxylation and oxygenation of ribulose 1,5-bisphosphate and hence to control the relative rates of photosynthetic carbon assimilation and photorespiration. Specificity factors of purified Rubisco from 24 species of C₃ plants found in diverse habitats with a wide range of environmental growth limitations by both water availability and temperature in the Balearic Islands were measured at 25 °C. The results suggest that specificity factors are more dependent on environmental pressure than on phylogenetic factors. Irrespective of phylogenetic relationships, higher specificity factors were found in species characteristically growing in dryer environments and in species that are hemideciduous or evergreen. Effects of temperature on specificity factor of the purified enzyme from 14 species were consistent with the concept that higher specificity factors were associated with an increase in the activation energy for oxygenation compared to carboxylation of the 2,3-enediolate of RuBP to the respective transition state intermediates. The results are discussed in terms of selection pressures leading to the differences in specificity factors and the value of the observations for identifying useful genetic manipulation to change Rubisco polypeptide subunits.     

Motif‐directed redesign of enzyme specificity

Computational protein design relies on several approximations, including the use of fixed backbones and rotamers, to reduce protein design to a computationally tractable problem. However, allowing backbone and off‐rotamer flexibility leads to more accurate designs and greater conformational diversity. Exhaustive sampling of this additional conformational space is challenging, and often impossible. Here, we report a computational method that utilizes a preselected library of native interactions to direct backbone flexibility to accommodate placement of these functional contacts. Using these native interaction modules, termed motifs, improves the likelihood that the interaction can be realized, provided that suitable backbone perturbations can be identified. Furthermore, it allows a directed search of the conformational space, reducing the sampling needed to find low energy conformations. We implemented the motif‐based design algorithm in Rosetta, and tested the efficacy of this method by redesigning the substrate specificity of methionine aminopeptidase. In summary, native enzymes have evolved to catalyze a wide range of chemical reactions with extraordinary specificity. Computational enzyme design seeks to generate novel chemical activities by altering the target substrates of these existing enzymes. We have implemented a novel approach to redesign the specificity of an enzyme and demonstrated its effectiveness on a model system.     

Regulation of Rubisco activity in vivo

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is not able to achieve and maintain adequate CO₂ and Mg²⁺ activation under physiological conditions. Higher plants and green algae contain Rubisco activase, a soluble protein which not only facilitates Rubisco activation in situ but also regulates enzyme activity in response to irradiance and other factors. Regulation of Rubisco activity by modulation of activation state coordinates the rate of CO₂ fixation with the rate of substrate regeneration. This regulation may be required to ensure that the levels of photosynthetic metabolites in the chloroplast are optimal for photosynthesis under a variety of environrmental conditions. Some plant species also appear to regulate Rubisco activity by synthesizing 2-carboxyarabinitol 1-phosphate, an inhibitor of Rubisco in the dark. This inhibitor may function primarily as a regulator of metabolite binding in the dark rather than as a modulator of Rubisco activity in the light.     

Structural basis for broad substrate specificity of earthworm fibrinolytic enzyme component A

Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1 pocket, which is typical for an elastase-like enzyme, but it can still hydrolyze varieties of substrates, and it exhibits wide substrate specificity. Former structure studies suggested that the four-residue insertion after Val(217) might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3A, we improved the native crystal structure to 1.8A and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9A. The final structures show that: (1) EFE-a possesses multisubstrate-binding sites interacting with the substrates; (2) significant conformation adjustment takes place at two loops binding to the N-terminal of the substrates, which may enhance the interaction between the enzyme and the substrates. These characteristics make the substrate-specificity of EFE-a less dependent on the property of its S1-pocket and may endow the enzyme with the ability to hydrolyze chymotrypsin-specific substrates and even trypsin-specific substrates.     

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.     

How to use the MEROPS database and website to help understand peptidase specificity

The MEROPS website ( https://www.ebi.ac.uk/merops ) and database was established in 1996 to present the classification and nomenclature of proteolytic enzymes. This was expanded to include a classification of protein inhibitors of proteolytic enzymes in 2004. Each peptidase or inhibitor is assigned to a distinct identifier, based on its biochemical and biological properties, and homologous sequences are assembled into a family. Families in which the proteins share similar tertiary structures are assembled into a clan. The MEROPS classification is thus a hierarchy with at least three levels (protein‐species, family, and clan) showing the evolutionary relationship. Several other data collections have been assembled, which are accessed from all levels in the hierarchy. These include, sequence homologs, selective bibliographies, substrate cleavage sites, peptidase–inhibitor interactions, alignments, and phylogenetic trees. The substrate cleavage collection has been assembled from the literature and includes physiological, pathological, and nonphysiological cleavages in proteins, peptides, and synthetic substrates. In this article, we make recommendations about how best to analyze these data and show analyses to indicate peptidase binding site preferences and exclusions. We also identify peptidases where co‐operative binding occurs between adjacent binding sites.     

Purification and specificity of two alpha-glucosidase isoforms of the parasitic protist Trichomonas vaginalis

Two isoforms of alpha-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.     

Sequence specificity of tRNA-modifying enzymes: An analysis of 258 tRNA sequences

The specificity and recognition of tRNA-modifying enzymes may be accounted for in part by nucleotide sequences which are localized next to the modifiable nucleoside. In order to determine the sequence specificity of tRNA-modifying enzymes, we have surveyed 55 published tRNA sequences from Escherichia coli, Salmonella typhimurium and T4 phage. For each modified nucleoside, the nucleotide sequence surrounding the modification site was determined for all tRNAs known to contain the modified nucleoside. Subsequently all tRNAs not containing the modified nucleoside were examined for the absence of the putative recognition site. We present the detailed analysis of 12 modified nucleosides for which we found a strong correlation between the modified nucleoside and the local nucleotide sequence. This suggests that these sequences may be recognition sites for tRNA-modifying enzymes. For each of the 12 modified nucleosides we have indentified a recognition sequence present in the tRNA set containing the modification and not in the set without it. All 203 other published tRNA sequences were then examined to see if the sequence specificity rules apply to other organisms, including both prokaryotes and eukaryotes. In several cases a good adherence was found, indicating conservation of the putative recognition sequences.     

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)