Buried Active Site Research Articles

Functional contacts for activation of urease from Helicobacter pylori: an integrated approach using evolutionary couplings, in-cell enzymatic assays, and computational docking

Introduction: Urease is an enzyme exploited by many virulent bacteria and fungi to infect the host and exert their virulence. The Gram-negative bacterium Helicobacter pylori relies on the activity of urease to infect the highly acidic human stomach. The activity of urease depends on the presence of a catalytic site containing two Ni(II) ions. In vivo, urease is initially synthesized as an inactive apo-enzyme and requires a post-translational activation process that involves the incorporation of the metal ions into its buried active site. In H. pylori, as well as in other bacteria, this activation process is mediated by four accessory proteins, named UreD, UreF, UreG, and UreE. Targeting the interactions between urease chaperones could potentially inhibit the activation of urease through blocking the Ni(II) ions incorporation, providing a route for the development of antimicrobial strategies against ureolytic pathogens.Methods: In this paper, an evolutionary couplings (EC) approach was adopted to determine the interaction surface between urease and UreD, the first protein that binds the enzyme, preparing it for the subsequent activation steps. Site-directed mutagenesis and an in-cell assay were used to detect urease activity in recombinant bacteria expressing the mutated operon. The obtained data were used to drive a protein-protein docking computational approach.Results and Discussion: The EC prediction retrieved ten pairs of residues lying at the interface between UreD and the urease subunit UreB, likely involved in contacts essential to build the protein complex. These contacts were largely confirmed experimentally, leading to the obtainment of a model for the urease-UreD complex that agrees well with the recently reported experimental cryo-EM structure. This work represents a proof of concept for the calculation of reliable models of protein interaction surfaces in the absence of experimental structures of critical assemblies.

Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity.

The haloalkane dehalogenase LinB is a well-known enzyme that contains buried active site and is used for many modelling studies. Using classical molecular dynamics simulations of enzymes and substrates, we searched for transient binding sites on the surface of the LinB protein by calculating maps of enzyme-ligand interactions that were then transformed into sparse matrices. All residues considered as functionally important for enzyme performance (e.g., tunnel entrances) were excluded from the analysis to concentrate rather on non-obvious surface residues. From a set of 130 surface residues, twenty-six were proposed as a promising improvement of enzyme performance. Eventually, based on rational selection and filtering out the potentially unstable mutants, a small library of ten mutants was proposed to validate the possibility of fine-tuning the LinB protein. Nearly half of the predicted mutant structures showed improved activity towards the selected substrates, which demonstrates that the proposed approach could be applied to identify non-obvious yet beneficial mutations for enzyme performance especially when obvious locations have already been explored.

In Silico Engineering of Enzyme Access Tunnels.

Enzyme engineering is a tailoring process that allows the modification of naturally occurring enzymes to provide them with improved catalytic efficiency, stability, or specificity. By introducing partial modifications to their sequence and to their structural features, enzyme engineering can transform natural enzymes into more efficient, specific and resistant biocatalysts and render them suitable for virtually countless industrial processes. Current enzyme engineering methods mostly target the active site of the enzyme, where the catalytic reaction takes place. Nonetheless, the tunnel that often connects the surface of an enzyme with its buried active site plays a key role in the activity of the enzyme as it acts as a gatekeeper and regulates the access of the substrate to the catalytic pocket. Hence, there is an increasing interest in targeting the sequence and the structure of substrate entrance tunnels in order to fine-tune enzymatic activity, regulate substrate specificity, or control reaction promiscuity.In this chapter, we describe the use of a rational in silico design and screening method to engineer the access tunnel of a fructosyl peptide oxidase with the aim to facilitate access to its catalytic site and to expand its substrate range. Our goal is to engineer this class of enzymes in order to utilize them for the direct detection of glycated proteins in diabetes monitoring devices. The design strategy involves remodeling of the backbone structure of the enzyme , a feature that is not possible with conventional enzyme engineering techniques such as single-point mutagenesis and that is highly unlikely to occur using a directed evolution approach.The proposed strategy, which results in a significant reduction in cost and time for the experimental production and characterization of candidate enzyme variants, represents a promising approach to the expedited identification of novel and improved enzymes. Rational enzyme design aims to provide in silico strategies for the fast, accurate, and inexpensive development of biocatalysts that can meet the needs of multiple industrial sectors, thus ultimately promoting the use of green chemistry and improving the efficiency of chemical processes.

Crystal structure of a key enzyme for anaerobic ethane activation.

Ethane, the second most abundant hydrocarbon gas in the seafloor, is efficiently oxidized by anaerobic archaea in syntrophy with sulfate-reducing bacteria. Here, we report the 0.99-angstrom-resolution structure of the proposed ethane-activating enzyme and describe the specific traits that distinguish it from methane-generating and -consuming methyl-coenzyme M reductases. The widened catalytic chamber, harboring a dimethylated nickel-containing F430 cofactor, would adapt the chemistry of methyl-coenzyme M reductases for a two-carbon substrate. A sulfur from methionine replaces the oxygen from a canonical glutamine as the nickel lower-axial ligand, a feature conserved in thermophilic ethanotrophs. Specific loop extensions, a four-helix bundle dilatation, and posttranslational methylations result in the formation of a 33-angstrom-long hydrophobic tunnel, which guides the ethane to the buried active site as confirmed with xenon pressurization experiments.

Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2–R1-dependent ribonucleotide reductase

Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport. After each catalytic cycle the active site contains a disulfide, which has to be reduced for turnover. Thioredoxin (Trx) and glutaredoxin (Grx) systems have been implicated as electron donors for the RNR disulfide reduction via the swinging arm. Using mouse R1-R2 and R1-p53R2 complexes, we found here that the catalytic efficiency of the GSH-Grx system is 4-6 times higher than that of the Trx1 system. For both complexes, the Vmax values for Grx are strongly depended on GSH concentrations. The GSH disulfide resulting from the Grx reaction was reduced by NADPH and GSH reductase and this enzyme was essential because reaction with GSH alone yielded only little activity. These results indicate that C-terminal shuttle dithiols of mammalian R1 have a crucial catalytic role and that the GSH-Grx system favors the R1-p53R2 enzyme for DNA replication in hypoxic conditions, mitochondrial DNA synthesis, and in DNA repair outside the S-phase.

Computational Study of Protein-Ligand Unbinding for Enzyme Engineering.

The computational prediction of unbinding rate constants is presently an emerging topic in drug design. However, the importance of predicting kinetic rates is not restricted to pharmaceutical applications. Many biotechnologically relevant enzymes have their efficiency limited by the binding of the substrates or the release of products. While aiming at improving the ability of our model enzyme haloalkane dehalogenase DhaA to degrade the persistent anthropogenic pollutant 1,2,3-trichloropropane (TCP), the DhaA31 mutant was discovered. This variant had a 32-fold improvement of the catalytic rate toward TCP, but the catalysis became rate-limited by the release of the 2,3-dichloropropan-1-ol (DCP) product from its buried active site. Here we present a computational study to estimate the unbinding rates of the products from DhaA and DhaA31. The metadynamics and adaptive sampling methods were used to predict the relative order of kinetic rates in the different systems, while the absolute values depended significantly on the conditions used (method, force field, and water model). Free energy calculations provided the energetic landscape of the unbinding process. A detailed analysis of the structural and energetic bottlenecks allowed the identification of the residues playing a key role during the release of DCP from DhaA31 via the main access tunnel. Some of these hot-spots could also be identified by the fast CaverDock tool for predicting the transport of ligands through tunnels. Targeting those hot-spots by mutagenesis should improve the unbinding rates of the DCP product and the overall catalytic efficiency with TCP.

Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer.

Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold-binding peptide at its amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine combinations of recombinant GDH proteins on the electrode show significantly different direct electron-transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme-electrode interface even in the absence of the electron-transfer subunit (i.e., β subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (-)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH2 redox wave and a stabilized bioelectrocatalytic current of more than 100 μA, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 μA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron-transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific "sticky-ended" proteins without loss of catalytic activity could be applied to other redox enzymes having a buried active site.

Conformational changes allow processing of bulky substrates by a haloalkane dehalogenase with a small and buried active site.

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.

Impact of the access tunnel engineering on catalysis is strictly ligand-specific.

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.

Role of an N-terminal extension in stability and catalytic activity of a hyperthermostable α/β hydrolase fold esterase.

The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical α/β hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of ~30-40°C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.

Steered Molecular Dynamic Simulations Reveal Critical Residues for (Un)Binding of Substrates, Inhibitors and a Product of the Malarial PFM1AAP

Malaria is one of the most prevalent and fatal infectious diseases of the world and is compounded by wide spread drug resistance, with some strains developing resistance to current front-line anti-malarials. Plasmodium falciparum M1 alanyl aminopeptidase (PFM1AAP) is involved in the terminal stages of haemoglobin degradation and the generation of an amino acid pool to support parasitic growth and development. This represents a promising new anti-malarial target where inhibition of this enzyme is lethal to the malarial parasite. To start understanding ligand recognition and binding in PFM1AAP for future drug design efforts, we explored (un)binding pathways of substrates, inhibitors and a product in the enzyme using steered molecular dynamics (sMD) simulations. The results of our simulations further clarify the substrate entrance and exit pathways to the buried active site of PFM1AAP and identify their respective physiochemical profiles. Our SMD simulations reveal several binding characteristics: (1) A substrate/inhibitor recognition mechanism, (2) active migration into the entrance channel, (3) a water reservoir which facilitates correct substrate orientation, (4) a molecular gate controlling product egress. Furthermore, several critical residues have been identified that in concert, facilitate these processes. These residues are now the focus of a mutagenesis study to fortify the in silico predictions. We observe differences in (un)binding pathways of substrates, inhibitors and product. A novel residue-based network analysis has been developed to highlight repetitive rather than sporadic events in the multiple sMD simulation trajectories of ligand (un)binding. Our work paves the way toward designing novel potent PFM1AAP inhibitors for the treatment of malaria.

Engineering a de Novo Transport Tunnel

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional “hole” into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (k...

Reversible Unfolding of Rhomboid Intramembrane Proteases

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.

Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes

BackgroundA cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. MethodsTime course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. ResultsThe global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. ConclusionsSize discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. General significanceCatalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site.

Allosteric regulation of rhomboid intramembrane proteolysis.

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P.stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

Catalytic Cycle of Penicillin Acylase from Escherichia coli: QM/MM Modeling of Chemical Transformations in the Enzyme Active Site upon Penicillin G Hydrolysis

Penicillin acylase from Escherichia coli is a unique enzyme that belongs to the recently discovered superfamily of N-terminal nucleophile hydrolases. It catalyzes selective hydrolysis of the side chain amide bond of penicillins and cephalosporins while leaving the labile amide bond in the β-lactam ring intact. Despite wide applications of penicillin acylase in the industry of β-lactam antibiotics and production of chiral amino compounds, its catalytic mechanism at atomic resolution has not yet been characterized. The complete cycle of chemical transformations of the most specific substrate of the enzyme, penicillin G, leading to formation of 6-aminopenicillanic and phenylacetic acids was modeled following quantum mechanics–molecular mechanics (QM/MM) calculations of the minimum energy reaction profile. The active site residues and the substrate were included in the QM part, and the rest of the system was treated applying molecular mechanics and classical force field parameters. The 3D structures in the enzyme active site corresponding to the noncovalent enzyme–substrate complex, the covalent acylenzyme intermediate, the noncovalent enzyme–product complex, the tetrahedral intermediates, and the respective transition states have been identified. QM/MM studies have shown that the α-amino group of the N-terminal catalytic βSer1 plays a key role in the catalytic machinery and directly assists its hydroxyl group in a proton relay at major stages of penicillin acylase catalytic mechanism, formation and hydrolysis of the covalent acylenzyme intermediate, which are characterized by close energy barriers. The βSer1 residue together with the oxyanion hole residues βAla69 and βAsn241 as well as βArg263 and βGln23 constitute a buried active site interaction network responsible for stabilization of tetrahedral intermediates, transition states, orientation of substrate and catalytic residues. βArg263 and βGln23 maintain the integrity of the catalytic machinery: βArg263 participates in orientation of the substrate as well as the α-amino group of βSer1 and coordinates the oxyanion hole residue βAsn241 across the whole catalytic cycle, whereas the backbone of βGln23 is responsible for orientation of both the βSer1 and the substrate. These results deliver insight into the earlier unknown ability of N-terminal amino acid to activate its own nucleophilic group directly as well as into organization of the stabilizing interaction network in penicillin acylase’s active site and will be used to design more effective enzyme variants for synthesis of new penicillins and cephalosporins.

Exploring Inhibitor Release Pathways in Histone Deacetylases Using Random Acceleration Molecular Dynamics Simulations

Molecular channel exploration perseveres to be the prominent solution for eliciting structure and accessibility of active site and other internal spaces of macromolecules. The volume and silhouette characterization of these channels provides answers for the issues of substrate access and ligand swapping between the obscured active site and the exterior of the protein. Histone deacetylases (HDACs) are metal-dependent enzymes that are involved in the cell growth, cell cycle regulation, and progression, and their deregulations have been linked with different types of cancers. Hence HDACs, especially the class I family, are widely recognized as the important cancer targets, and the characterizations of their structures and functions have been of special interest in cancer drug discovery. The class I HDACs are known to possess two different protein channels, an 11 Å and a 14 Å (named channels A and B1, respectively), of which the former is a ligand or substrate occupying tunnel that leads to the buried active site zinc ion and the latter is speculated to be involved in product release. In this work, we have carried out random acceleration molecular dynamics (RAMD) simulations coupled with the classical molecular dynamics to explore the release of the ligand, N-(2-aminophenyl) benzamide (LLX) from the active sites of the recently solved X-ray crystal structure of HDAC2 and the computationally modeled HDAC1 proteins. The RAMD simulations identified significant structural and dynamic features of the HDAC channels, especially the key 'gate-keeping' amino acid residues that control these channels and the ligand release events. Further, this study identified a novel and unique channel B2, a subchannel from channel B1, in the HDAC1 protein structure. The roles of water molecules in the LLX release from the HDAC1 and HDAC2 enzymes are also discussed. Such structural and dynamic properties of the HDAC protein channels that govern the ligand escape reactions will provide further mechanistic insights into the HDAC enzymes, which, in the long run, have a potential to bring new ideas for developing more promising HDAC inhibitors as well as extend our atomic level understandings on their mechanisms of action.

Modulating O2 Reactivity in a Fungal Flavoenzyme: INVOLVEMENT OF ARYL-ALCOHOL OXIDASE PHE-501 CONTIGUOUS TO CATALYTIC HISTIDINE

Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O(2) to H(2)O(2) in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O(2) access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O(2) reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ∼2-fold that of native AAO and ∼120-fold that of F501A, seems related to a higher O(2) availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O(2) inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O(2) population at 3-4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O(2) reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O(2) at an adequate distance from flavin C4a (and His-502 Nε). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O(2) reactivity of this flavoenzyme.

Ribonuclease J: How to Lead a Double Life

New structures of RNase J reported by Dorleans et al. and Newman et al. in this issue of Structure suggest how an enzyme whose identical subunits each contain a single buried active site can function as both a 5′ exonuclease and an endonuclease.

Diffusion-influenced ligand binding to buried sites in macromolecules and transmembrane channels

We consider diffusion-influenced binding to a buried binding site that is connected to the surface by a narrow tunnel. Under the single assumption of an equilibrium distribution of ligands over the tunnel cross section, we reduce the calculation of the time-dependent rate coefficient to the solution of a one-dimensional diffusion equation with appropriate boundary conditions. We obtain a simple analytical expression for the steady-state rate that depends on the potential of mean force in the tunnel and the diffusion-controlled rate of binding to the tunnel entrance. Potential applications of our theory include substrate binding to a buried active site of an enzyme and permeant ion binding to an internal site in a transmembrane channel.