Structure-function Relationships Of Enzymes Research Articles

Elucidating the Enzymatic Mechanism of Dihydrocoumarin Degradation: Insight into the Functional Evolution of Methyl-Parathion Hydrolase from QM/MM and MM MD Simulations.

Methyl-parathion hydrolase (MPH), which evolved from dihydrocoumarin hydrolase, offers one of the most efficient enzymes for the hydrolysis of methyl-parathion. Interestingly, the substrate preference of MPH shifts from the methyl-parathion to the lactone dihydrocoumarin (DHC) after its mutation of five specific residues (R72L, L273F, L258H, T271I, and S193Δ, m5-MPH). Here, extensive QM/MM calculations and MM MD simulations have been used to delve into the structure-function relationship of MPH enzymes and plausible mechanisms for the chemical and nonchemical steps, including the transportation and binding of the substrate DHC to the active site, the hydrolysis reaction, and the product release. The results reveal that the five mutations remodel the active pocket and reposition DHC within the active site, leading to stronger enzyme-substrate interactions. The MM/GBSA-estimated binding free energies are about -20.7 kcal/mol for m5-MPH and -17.1 kcal/mol for wild-type MPH. Furthermore, this conformational adjustment of the protein may facilitate the chemical step of DHC hydrolysis and the product release, although there is a certain influence on the substrate transport. The hydrolytic reaction begins with the nucleophilic attack of the bridging OH- with the energy barriers of 22.0 and 18.0 kcal/mol for the wild-type and m5-MPH enzymes, respectively, which is rate-determining for the entire process. Unraveling these mechanistic intricacies may help in the understanding of the natural evolution of enzymes for diverse substrates and establish the enzyme structure-function relationship.

ICEED: Integrated customized extraction of enzyme data.

Enzymes catalyze diverse biochemical reactions and are building blocks of cellular and metabolic pathways. Data and metadata of enzymes are distributed across databases and are archived in various formats. The enzyme databases provide utilities for efficient searches and downloading enzyme records in batch mode but do not support organism-specific extraction of subsets of data. Users are required to write scripts for parsing entries for customized data extraction prior to downstream analysis. Integrated Customized Extraction of Enzyme Data (iCEED) has been developed to provide organism-specific customized data extraction utilities for seven commonly used enzyme databases and brings these resources under an integrated portal. iCEED provides dropdown menus and search boxes using typehead utility for submission of queries as well as enzyme class-based browsing utility. A utility to facilitate mapping and visualization of functionally important features on the three-dimensional (3D) structures of enzymes is integrated. The customized data extraction utilities provided in iCEED are expected to be useful for biochemists, biotechnologists, computational biologists, and life science researchers to build curated datasets of their choice through an easy to navigate web-based interface. The integrated feature visualization system is useful for a fine-grained understanding of the enzyme structure-function relationship. Desired subsets of data, extracted and curated using iCEED can be subsequently used for downstream processing, analyses, and knowledge discovery. iCEED can also be used for training and teaching purposes.

Thermophilic lipases in industrial applications: Stutzerimonas stutzeri as a Case Study in Nigeria and the US

Thermophilic lipases derived from Stutzerimonas stutzeri have garnered significant attention in recent years due to their robustness and efficiency in various industrial applications. This review delves into the research, development, and application of these enzymes, with a comparative analysis between Nigeria and the United States. In both nations, researchers have explored the enzymatic properties of thermophilic lipases from Stutzerimonas stutzeri, aiming to optimize their performance for diverse industrial processes. The research encompasses studies on enzyme purification, characterization, and genetic engineering to enhance catalytic activity and stability under extreme conditions. Additionally, efforts have been directed towards understanding the enzyme's structure-function relationship to tailor it for specific applications. In Nigeria, the focus on thermophilic lipases from Stutzerimonas stutzeri has been primarily driven by the need for sustainable biotechnological solutions in various industries, including food, pharmaceuticals, and biofuel production. The enzymes' thermostability makes them particularly suitable for high-temperature processes prevalent in Nigeria's tropical climate, offering potential for cost-effective and eco-friendly alternatives to conventional chemical catalysts. In contrast, the United States has witnessed extensive research and development in thermophilic lipases for industrial applications, with a strong emphasis on biocatalysis in the pharmaceutical, detergent, and biofuel sectors. The advanced infrastructure and investment in biotechnology have facilitated the commercialization of Stutzerimonas stutzeri lipases, leading to their integration into various industrial processes to improve efficiency and sustainability. Despite the similarities in research objectives, disparities exist in the technological advancements and industrial utilization of thermophilic lipases from Stutzerimonas stutzeri between Nigeria and the US. While Nigeria faces challenges related to limited resources and infrastructure, the US benefits from well-established research institutions and industrial networks, enabling accelerated innovation and commercialization. Bridging these gaps through international collaborations and knowledge exchange could facilitate the widespread adoption of thermophilic lipases for sustainable industrial development globally.

Development of a fluorescence-based excipient screening for improved stability and shelf-life of recombinant chitin deacetylase

Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase (BaCDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating BaCDA stability with its activity. The optimised formulation comprises 50 mM Tris-HCl buffer pH 7, 1 M NaCl, 20 % glycerol, and 1 mM Mg2+ as excipients. This formulation significantly improves BaCDA's thermostability (140.47 % increase) and enzyme activity (2.9-fold enhancement). BaCDA remains stable in the formulated buffer at −20 °C and −80 °C for 30 days and at 4 °C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.

On the interface of enzyme and spatial confinement: The impacts of confinement rigidity, shape, and surface properties on the interplay of enzyme structure, dynamics, and function

Confining proteins in synthetic nanoscale spatial compartments has offered a cell-free avenue to understand enzyme structure–function relationships and complex cellular processes near the physiological conditions, an important branch of fundamental protein biophysics studies. Enzyme confinement has also provided advancement in biocatalysis by offering enhanced enzyme reusability, cost-efficiency, and substrate selectivity in certain cases for research and industrial applications. However, the primary research efforts in this area have been focused on the development of novel confinement materials and investigating protein adsorption/interaction with various surfaces, leaving a fundamental knowledge gap, namely, the lack of understanding of the confined enzymes (note that enzyme adsorption to or interactions with surfaces differs from enzyme confinement as the latter offers an enhanced extent of restriction to enzyme movement and/or conformational flexibility). In particular, there is limited understanding of enzymes' structure, dynamics, translocation (into biological pores), folding, and aggregation in extreme cases upon confinement, and how confinement properties such as the size, shape, and rigidity affect these details. The first barrier to bridge this gap is the difficulty in “penetrating” the “shielding” of the confinement walls experimentally; confinement could also lead to high heterogeneity and dynamics in the entrapped enzymes, challenging most protein-probing experimental techniques. The complexity is raised by the variety in the possible confinement environments that enzymes may encounter in nature or on lab benches, which can be categorized to rigid confinement with regular shapes, rigid restriction without regular shapes, and flexible/dynamic confinement which also introduces crowding effects. Thus, to bridge such a knowledge gap, it is critical to combine advanced materials and cutting-edge techniques to re-create the various confinement conditions and understand enzymes therein. We have spearheaded in this challenging area by creating various confinement conditions to restrict enzymes while exploring experimental techniques to understand enzyme behaviors upon confinement at the molecular/residue level. This review is to summarize our key findings on the molecular level details of enzymes confined in (i) rigid compartments with regular shapes based on pre-formed, mesoporous nanoparticles and Metal–Organic Frameworks/Covalent-Organic Frameworks (MOFs/COFs), (ii) rigid confinement with irregular crystal defects with shapes close to the outline of the confined enzymes via co-crystallization of enzymes with certain metal ions and ligands in the aqueous phase (biomineralization), and (iii) flexible, dynamic confinement created by protein-friendly polymeric materials and assemblies. Under each case, we will focus our discussion on (a) the way to load enzymes into the confined spaces, (b) the structural basis of the function and behavior of enzymes within each compartment environments, and (c) technical advances of our methodology to probe the needed structural information. The purposes are to depict the chemical physics details of enzymes at the challenging interface of natural molecules and synthetic compartment materials, guide the selection of enzyme confinement platforms for various applications, and generate excitement in the community on combining cutting-edge technologies and synthetic materials to better understand enzyme performance in biophysics, biocatalysis, and biomedical applications.

Site-directed spin labeling-electron paramagnetic resonance spectroscopy in biocatalysis: Enzyme orientation and dynamics in nanoscale confinement

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy probes the otherwise inaccessible structural information in complex biological systems. We recently extended SDSL-EPR to reveal the relative orientation and backbone dynamics of enzymes upon encapsulation in mesoporous nanostructures, which set the structural basis underlying the observed biocatalytic activity. Our strategy had generated interest in the biocatalysis community, and thus in this resource article, we contribute an introduction to the principles and experimental procedure that generalize SDSL-EPR to heterogeneous biocatalysis. We will focus on enzymes in mesoporous materials with examples demonstrating the methods and cautions of potential pitfalls. The ultimate goal is to provide the biocatalysis community with a powerful resource to fill in a long-standing knowledge gap in heterogeneous biocatalysis and the structure-function relationship of enzymes at the interface of enzyme-mesoporous materials and utilize the structural insights to guide the rational design of porous platforms for enzyme immobilization. • Enzyme orientation is determined by using EPR to probe its contact with nanoscale pores • Enzyme dynamics in nanoscale pores and associated catalytic activities are revealed • Protocols and procedures are given for experimental data acquisition, analysis, and interpretation • Various EPR techniques and spin labels can be extended to heterogeneous biocatalysis Enzyme immobilization in nanoscale confinement offers enhanced enzyme protection and reusability, yet the structure-function relationship of the entrapped enzymes remains elusive. Site-directed spin labeling in combination with electron paramagnetic resonance (EPR) spectroscopy has primarily been applied in determining protein structural information in complex biological systems. In this resource article, we introduce the extension of the X-band continuous-wave EPR with a methanethiosulfonate spin label to reveal enzyme structural basis upon immobilization into nanoscale confinement. Using our recent works as examples, we demonstrate the method for determining enzyme orientation and backbone dynamics upon immobilization with a special focus on generalizing the method to heterogeneous biocatalysis and cautions in each step. Furthermore, we summarize the recent findings in other EPR techniques and spin labels so that the whole EPR toolbox can be extended to heterogeneous biocatalysis. In this resource article, Yang and colleagues introduce a general procedure that can reveal the orientation and backbone dynamics of enzymes entrapped in mesoporous materials based on metal-organic materials (MOMs) by using X-band SDSL-EPR. They demonstrate the method by using recently published works focusing on enzyme-MOM interfaces, as well as highlight cautions and potential solutions at each step. They then summarize a variety of other EPR techniques and spin labels in order to pinpoint their potential applications in heterogeneous biocatalysis in the near future.

Functional and structural characterization of a novel GH3 β-glucosidase from the gut metagenome of the Brazilian Cerrado termite Syntermes wheeleri

In this study, a GH3 family β-glucosidase (Bgl7226) from metagenomic sequences of the Syntermes wheeleri gut, a Brazilian Cerrado termite, was expressed, purified and characterized. The enzyme showed two optimum pHs (pH 7 and pH 10), and a maximum optimum temperature of about 40 °C using 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrate. Bgl7226 showed higher enzymatic activity at basic pH, but higher affinity (Km) at neutral pH. However, at neutral pH the Bgl7226 enzyme showed higher catalytic efficiency (kcat/Km) for pNPG substrate. Predictive analysis about the enzyme structure-function relationship by sequence alignment suggested the presence of multi-domains and conserved catalytic sites. Circular dichroism results showed that the secondary structure composition of the enzyme is pH-dependent. Small conformational changes occurred close to the optimum temperature of 40 o C, and seem important for the highest activity of Bgl7226 observed at pH 7 and 10. In addition, the small transition in the unfolding curves close to 40 o C is typical of intermediates associated with proteins structured in several domains. Bgl7226 has significant β-glucosidase activity which could be attractive for biotechnological applications, such as plant roots detoxification; specifically, our group is interested in cassava roots (Manihot esculenta) detoxification.

Point mutations in the different domains of the Saccharomyces cerevisiae plasma membrane PMA1 ATPase cause redistribution among fractions of inorganic polyphosphates

Both ATP and inorganic polyphosphates (PolyP) appeared to be involved in the yeast energy homeostasis, in which plasma membrane PMA1 H+-АТРase plays one of the key roles. During biogenesis and functioning, the enzyme undergoes structural and regulatory phosphorylation. Aim of the work was to elucidate interconnection between functioning of the yeast PMA1 H+-АТРase carrying point substitutions that affected the enzyme structure-function relationship and its ability to be phosphorylated and PolyP metabolism. Effect of such replacements of phosphorylable and non-phosphorylable residues in three topologically and functionally different domains of the enzyme – membrane, extracytosolic, and C-terminal – on the metabolism of polyphosphates and distribution between short-, mid-, and long-chained PolyP fractions (PolyP1-PolyP4-5) has been studied. АТРase activity of membrane and most extracytosolic strains was noticeably lower comparing to the wild type. Of these mutants, three substitutions (L801A, E803A, E847A) have not caused significant changes in PolyP content regardless up to twofold drop of the ATPase activity; F796A with four-fold decreased activity has led to noticeable increase of mid-chained PolyP fractions. The most pronounced effect of PolyP redistribution was caused either by removal of potential (S846A, T850A, D851A) or established (S911A) phosphosites in the PMA1 ATPase or by altering type of the established phosphosite (S911D, T912D). Patterns of PolyP fractions for these two groups have significantly differed from each other, occurring in opposite directions for mutants with removed and changed phosphosite. Changing residue of phosphosite without altering its type (T850S) has not led to significant changes in PolyP content. Communicated by Ramaswamy H. Sarma

Proteins-Based Nanocatalysts for Energy Conversion Reactions.

In recent years, the incorporation of molecular enzymes into nanostructured frameworks to create efficient energy conversion biomaterials has gained increasing interest as a promising strategy owing to both the dynamic behavior of proteins for their electrocatalytic function and the unique properties of the synergistic interactions between proteins and nanosized materials. Herein, we review the impact of proteins on energy conversion fields and the contribution of proteins to the improved activity of the resulting nanocomposites. We address different strategies to fabricate protein-based nanocatalysts as well as current knowledge on the structure-function relationships of enzymes during the catalytic processes. Additionally, a comprehensive review of state-of-the-art bioelectrocatalytic materials for water-splitting reactions such as hydrogen evolution reaction (HER) and oxygen evolution reactions (OER) is afforded. Finally, we briefly envision opportunities to develop a new generation of electrocatalysts towards the electrochemical reduction of N2 to NH3 using theoretical tools to built nature-inspired nitrogen reduction reaction catalysts.

Structure–function relationships of radical SAM enzymes

Radical S-adenosyl-l-methionine (SAM) enzymes belong to a family of catalysts whose number of annotated sequences is still growing. Upon the one-electron reduction of a [Fe4S4] cluster, they can cleave SAM to produce a highly reactive 5′-deoxyadenosyl radical species. This radical species in turn triggers a wide variety of radical-based reactions on substrates ranging from small organic molecules to proteins, DNA or RNA. The challenging reactions they catalyse makes them very promising catalysts for diverse biotechnological applications. However, the high-energy intermediates involved require fine control of the chemistry by the protein matrix. Understanding their control mechanism is a prerequisite for a broader use of these enzymes as synthetic tools. Here I review some of the latest developments in the field, focusing on the structure–function relationship of a few examples for which three-dimensional structures, in vitro and spectroscopic data, as well as theoretical calculations, are available to better describe the close interaction between the chemistry performed and the tight control of the protein matrix. Radical S-adenosyl-l-methionine enzymes catalyse challenging chemical reactions, showing potential for biotechnological applications. This Review focuses on enzyme structure–function relationships, providing insights on how these biocatalysts perform different reactions and control the reactive radical species.

Visualizing ubiquitination in mammalian cells.

Covalent modification of proteins with ubiquitin is essential for the majority of biological processes in mammalian cells. Numerous proteins are conjugated with single or multiple ubiquitin molecules or chains in a dynamic fashion, often determining protein half-lives, localization or function. Experimental approaches to study ubiquitination have been dominated by genetic and biochemical analysis of enzyme structure-function relationships, reaction mechanisms and physiological relevance. Here, we provide an overview of recent developments in microscopy-based imaging of ubiquitination, available reagents and technologies. We discuss the progress in direct and indirect imaging of differentially linked ubiquitin chains in fixed and living cells using confocal fluorescence microscopy and super-resolution microscopy, illustrated by the role of ubiquitin in antibacterial autophagy and pro-inflammatory signalling. Finally, we speculate on future developments and forecast a transition from qualitative to quantitative super-resolution approaches to understand fundamental aspects of ubiquitination and the formation and distribution of functional E3 ligase protein complexes in their native environment.

Effect of the hyaluronidase microe nvironment on the enzyme structure–function relationship and computational study of the in silico molecular docking of chondroitin sulfate and heparin short fragments to hyaluronidase

The review addresses the biochemical interactions of hyaluronidases with components of the natural microenvironment. The effect of subtle structural differences between ligands on the enzyme structure–function relationship regulation is noted. Docking of chondroitin sulfate (CS) trimers (hexasaccharides) and heparin tetramers (octasaccharides) to the 3D model of the bovine testicular hyaluronidase (BTH) was performed by computational chemistry methods in order to elucidate the mechanism of regulation of the enzyme functioning in the body (using virtual screening, molecular dynamics, and calculation of surface electrostatic potential of protein complexes). Several binding sites for glycosaminoglycan (GAG) ligands were found to occur on the hyaluronidase surface. They are identical for CS trimers and heparin tetramers. The calculations showed the possibility of both reversible and irreversible conformational changes of the 3D structure of BTH, depending on the arrangement of negatively charged ligands on its globule. When the changes are irreversible, Glu-149 and Asp-147, which are key amino acid residues for the catalytic activity of BTH, can migrate from the vicinity of the native enzyme active site to the periphery of the protein molecule, thus inducing enzyme inactivation. The interaction of the GAG ligands with the BTH active site is mainly caused by electrostatic forces. Four or five binding sites of the chondroitin sulfate trimer proved to be critical for stabilization of the enzyme structure. Their occupation was sufficient for preventing irreversible deformation of the BTH molecule upon the insertion of the heparin ligand into the active site cavity. Protein stabilization is accompanied by the formation of a particular form of the surface electrostatic potential.

Large-scale QM/MM free energy simulations of enzyme catalysis reveal the influence of charge transfer.

Hybrid quantum mechanical-molecular mechanical (QM/MM) simulations provide key insights into enzyme structure-function relationships. Numerous studies have demonstrated that large QM regions are needed to systematically converge ground state, zero temperature properties with electrostatic embedding QM/MM. However, it is not well known if ab initio QM/MM free energy simulations have this same dependence, in part due to the hundreds of thousands of energy evaluations required for free energy estimations that in turn limit QM region size. Here, we leverage recent advances in electronic structure efficiency and accuracy to carry out range-separated hybrid density functional theory free energy simulations in a representative methyltransferase. By studying 200 ps of ab initio QM/MM dynamics for each of five QM regions from minimal (64 atoms) to one-sixth of the protein (544 atoms), we identify critical differences between large and small QM region QM/MM in charge transfer between substrates and active site residues as well as in geometric structure and dynamics that coincide with differences in predicted free energy barriers. Distinct geometric and electronic structure features in the largest QM region indicate that important aspects of enzymatic rate enhancement in methyltransferases are identified with large-scale electronic structure.

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous substrates such as O2, H2, and N2 This work investigates the role of a defined pathway for O2 in regulating the peroxidation of linoleic acid by soybean lipoxygenase 1. Computational and mutagenesis studies provide strong support for a dominant delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio- and stereospecificity of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas migration pathway. The findings show that a single site mutation (I553W) limits oxygen accessibility to the active site, greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site mutation (L496W) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio- and stereospecificity of peroxidation. The present data indicate that modest changes in a protein scaffold may modulate the access of small gaseous molecules to enzyme-bound substrates.

Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance.

Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.

Recent Progress in the Discovery of Next Generation Inhibitors of Aromatase from the Structure-Function Perspective.

Human aromatase catalyzes the synthesis of estrogen from androgen with high substrate specificity. For the past 40 years, aromatase has been a target of intense inhibitor discovery research for the prevention and treatment of estrogen-dependent breast cancer. The so-called third generation aromatase inhibitors (AIs) letrozole, anastrozole, and the steroidal exemestane were approved in the U.S. in the late 1990s for estrogen-dependent postmenopausal breast cancer. Efforts to develop better AIs with higher selectivity and lower side effects were handicapped by the lack of an experimental structure of this unique P450. The year 2009 marked the publication of the crystal structure of aromatase purified from human placenta, revealing an androgen-specific active site. The structure has reinvigorated research activities on this fascinating enzyme and served as the catalyst for next generation AI discovery research. Here, we present an account of recent developments in the AI field from the perspective of the enzyme's structure-function relationships.

A comprehensive alanine-scanning mutagenesis study reveals roles for salt bridges in the structure and activity of Pseudomonas aeruginosa elastase.

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k cat/K m), half-lives (t 1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.

191 Point mutations in the yeast Pma1 H+-ATPase affect polyphosphate (PolyP) distribution

Yeast plasma membrane Н+-ATPase (Pma1) is a key enzyme of the yeast cell metabolism. It generates electrochemical Н+ gradient providing energy for operating the secondary solute transport systems and maintaining intracellular pH and ion homeostasis. Most of the enzyme molecule anchored in the plasma membrane by M1–M10 segments is located in cytosole and membrane; less than 5% of the Pma1 face extracellular space. Membrane domain contains amino acid residues, which form H+ transport pathway; cytosole parts house the enzyme active center and cytosolic C-terminal tail has regulatory function. The enzyme function and regulation are tightly connected to glucose metabolism: its fermentation triggers activation of Pma1 function, structurally accompanied by the enzyme multiple phosphorylation during intracellular traffic on route to plasma membrane. There are ca. 10 phosphorylation sites; only 3 of them are identified: one single and two tandemly located sites are in the C-terminal tail. Both ATP and PolyP can be used to phosphorylate amino acid residues; however, there are little data on the interactive metabolism of ATP and PolyP. Most of phosphorylable Ser, Thr, Asp, Glu, and Tyr residues are located in the inner parts of the enzyme; however, there are several such residues in the Pma1 outer parts: D714, S716, D718, and D720 in M5–M6 loop and S846, E847, T850, and D851 in M9–M10 loop, close to the enzyme regulatory C-tail. It seems reasonable that multiple phosphorylation of Pma1 goes subsequently, and first of such sites could be located in the enzyme extracytosolic part. The M5–M6 loop phosphorylable residues, except D714, were found to be unimportant for the enzyme structure-function relationship; D714A mutant was poorly expressed and inactive (Petrov, 2011). However, D714N did not disturb the enzyme functioning, thus excluding the role of D714 in the enzyme phosphorylation. Therefore, we choose to study further residues in the M9–M10 loop by replacing them with Ala. The ATPase activity of these mutants ranged from the wild-type level (S846A) to 2- (E847A) to 3-fold (T850A) drop. Changes of activity were accompanied by changes in PolyP fractions (Figure 1), which were most significant for S846A and T850A. S846A had 1.5–1.7-fold increase in PolyP1 (found mostly in cytosole and vacuoles) and PolyP3 (localized near the cell surface) and dramatic 3-fold decrease in PolyP4-5 fractions (associated with cell wall), while T850A had stable 1.5-fold increase in all PolyP but PolyP4-5 fractions. Both mutants also had 20% (S846A) to 37% (T850A) increase in total PolyP. These data may point to lack of one or more phosphorylation sites and/or participation of PolyP in the Pma1 ATPase phosphorylation. Possibly, the sites at S846 and T850 act jointly, similarly to tandemly located and acting S911 and T912 in the regulatory C-tail (Lecchi et al., 2007). Further study of these mutants, although methodologically challenging, seems certain to yield more useful insights into functioning and regulation of the Pma1 ATPase as well as into the interactive mechanisms of ATP and PolyP metabolism.

Structure–function relationships in calpains

Calpains are a family of complex multi-domain intracellular enzymes that share a calcium-dependent cysteine protease core. These are not degradative enzymes, but instead carry out limited cleavage of target proteins in response to calcium signalling. Selective cutting of cytoskeletal proteins to facilitate cell migration is one such function. The two most abundant and extensively studied members of this family in mammals, calpains 1 and 2, are heterodimers of an isoform-specific 80kDa large subunit and a common 28kDa small subunit. Structures of calpain-2, both Ca2+-free and bound to calpastatin in the activated Ca2+-bound state, have provided a wealth of information about the enzyme's structure-function relationships and activation. The main association between the subunits is the pairing of their C-terminal penta-EF-hand domains through extensive intimate hydrophobic contacts. A lesser contact is made between the N-terminal anchor helix of the large subunit and the penta-EF-hand domain of the small subunit. Up to ten Ca2+ ions are co-operatively bound during activation. The anchor helix is released and individual domains change their positions relative to each other to properly align the active site. Because calpains 1 and 2 require ~30 and ~350μM Ca2+ ions for half-maximal activation respectively, it has long been argued that autoproteolysis, subunit dissociation, post-translational modifications or auxiliary proteins are needed to activate the enzymes in the cell, where Ca2+ levels are in the nanomolar range. In the absence of robust support for these mechanisms, it is possible that under normal conditions calpains are transiently activated by high Ca2+ concentrations in the microenvironment of a Ca2+ influx, and then return to an inactive state ready for reactivation.

Structure–function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure–function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the 14C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer.