Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor.

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAalavia conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

ADR3, a next generation i-body to human RANKL, inhibits osteoclast formation and bone resorption.

Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue and puts tens of millions of people at high risk of fractures. New therapeutic agents like i-bodies, a class of next-generation single-domain antibodies, are needed to overcome some limitations of conventional treatments. An i-body is a human immunoglobulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies, the variable new antigen receptors of sharks. Its small size (∼12 kDa) and long binding loops provide access to drug targets, which are considered undruggable by traditional monoclonal antibodies. Here, we have successfully identified a human receptor activator of nuclear factor-κB ligand (RANKL) i-body, ADR3, which demonstrates a high binding affinity to human RANKL (hRANKL) with no adverse effect on the survival or proliferation of bone marrow-derived macrophages. Differential scanning fluorimetry suggested that ADR3 is stable and able to tolerate a wide range of physical environments (including both temperature and pH). In addition, in vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentiation, podosome belt formation, and bone resorption activity. Further investigation on the mechanism of action of ADR3 revealed that it can inhibit hRANKL-mediated signaling pathways, supporting the in vitro functional observations. These clues collectively indicate that hRANKL antagonist ADR3 attenuates osteoclast differentiation and bone resorption, with the potential to serve as a novel therapeutic to protect against bone loss.

N-acetyl-ß-hexosaminidase activity is important for chitooligosaccharide metabolism and biofilm formation in Burkholderia pseudomallei.

Burkholderia pseudomallei is a saprophytic Gram-negative bacillus that can cause the disease melioidosis. Although B. pseudomallei is a recognised member of terrestrial soil microbiomes, little is known about its contribution to the saprophytic degradation of polysaccharides within its niche. For example, while chitin is predicted to be abundant within terrestrial soils the chitinolytic capacity of B. pseudomallei is yet to be defined. This study identifies and characterises a putative glycoside hydrolase, bpsl0500, which is expressed by B. pseudomallei K96243. Recombinant BPSL0500 was found to exhibit activity against substrate analogues and GlcNAc disaccharides relevant to chitinolytic N-acetyl-ß-d-hexosaminidases. In B. pseudomallei, bpsl0500 was found to be essential for both N-acetyl-ß-d-hexosaminidase activity and chitooligosaccharide metabolism. Furthermore, bpsl0500 was also observed to significantly affect biofilm deposition. These observations led to the identification of BPSL0500 activity against model disaccharide linkages that are present in biofilm exopolysaccharides, a feature that has not yet been described for chitinolytic enzymes. The results in this study indicate that chitinolytic N-acetyl-ß-d-hexosaminidases like bpsl0500 may facilitate biofilm disruption as well as chitin assimilation, providing dual functionality for saprophytic bacteria such as B. pseudomallei within the competitive soil microbiome.

High-pressure single-crystal diffraction at the Australian Synchrotron.

A new high-pressure single-crystal diffraction setup has been designed and implemented at the Australian Synchrotron for collecting molecular and protein crystal structures. The setup incorporates a modified micro-Merrill-Bassett cell and holder designed specifically to fit onto the horizontal air-bearing goniometer, allowing high-pressure diffraction measurements to be collected with little to no modification of the beamline setup compared with ambient data collections. Compression data for the amino acid, L-threonine, and the protein, hen egg-white lysozyme, were collected, showcasing the capabilities of the setup.

Development of a novel spatiotemporal depletion system for cellular cholesterol.

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.

Novel small molecules that increase the susceptibility of Neisseria gonorrhoeae to cationic antimicrobial peptides by inhibiting lipid A phosphoethanolamine transferase.

OBJECTIVES: Neisseria gonorrhoeae is an exclusively human pathogen that commonly infects the urogenital tract resulting in gonorrhoea. Empirical treatment of gonorrhoea with antibiotics has led to multidrug resistance and the need for new therapeutics. Inactivation of lipooligosaccharide phosphoethanolamine transferase A (EptA), which attaches phosphoethanolamine to lipid A, results in attenuation of the pathogen in infection models. Small molecules that inhibit EptA are predicted to enhance natural clearance of gonococci via the human innate immune response. METHODS: A library of small-fragment compounds was tested for the ability to enhance susceptibility of the reference strain N. gonorrhoeae FA1090 to polymyxin B. The effect of these compounds on lipid A synthesis and viability in models of infection were tested. RESULTS: Three compounds, 135, 136 and 137, enhanced susceptibility of strain FA1090 to polymyxin B by 4-fold. Pre-treatment of bacterial cells with all three compounds resulted in enhanced killing by macrophages. Only lipid A from bacterial cells exposed to compound 137 showed a 17% reduction in the level of decoration of lipid A with phosphoethanolamine by MALDI-TOF MS analysis and reduced stimulation of cytokine responses in THP-1 cells. Binding of 137 occurred with higher affinity to purified EptA than the starting material, as determined by 1D saturation transfer difference NMR. Treatment of eight MDR strains with 137 increased susceptibility to polymyxin B in all cases. CONCLUSIONS: Small molecules have been designed that bind to EptA, inhibit addition of phosphoethanolamine to lipid A and can sensitize N. gonorrhoeae to killing by macrophages.

Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

A missense mutation sheds light on a novel structure-function relationship of RANKL.

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding.

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.

Computational site-directed mutagenesis studies of the role of the hydrophobic triad on substrate binding in cholesterol oxidase.

Cholesterol oxidase (ChOx) is a flavoenzyme that oxidizes and isomerizes cholesterol (CHL) to form cholest-4-en-3-one. Molecular docking and molecular dynamics simulations were conducted to predict the binding interactions of CHL in the active site. Several key interactions (E361-CHL, N485-FAD, and H447-CHL) were identified and which are likely to determine the correct positioning of CHL relative to flavin-adenine dinucleotide (FAD). Binding of CHL also induced changes in key residues of the active site leading to the closure of the oxygen channel. A group of residues, Y107, F444, and Y446, known as the hydrophobic triad, are believed to affect the binding of CHL in the active site. Computational site-directed mutagenesis of these residues revealed that their mutation affects the conformations of key residues in the active site, leading to non-optimal binding of CHL and to changes in the structure of the oxygen channel, all of which are likely to reduce the catalytic efficiency of ChOx. Proteins 2017; 85:1645-1655. © 2017 Wiley Periodicals, Inc.

Direct demonstration of lipid phosphorylation in the lipid bilayer of the biomimetic bicontinuous cubic phase using the confined enzyme lipid A phosphoethanolamine transferase.

Retention of amphiphilic protein activity within the lipid bilayer membrane of the nanostructured biomimetic bicontinuous cubic phase is crucial for applications utilizing these hybrid protein-lipid self-assembly materials, such as in meso membrane protein crystallization and drug delivery. Previous work, mainly on soluble and membrane-associated enzymes, has shown that enzyme activity may be modified when immobilized, including membrane bound enzymes. The effect on activity may be even greater for amphiphilic enzymes with a large hydrophilic domain, such as the Neisserial enzyme lipid A phosphoethanolamine transferase (EptA). Encapsulation within the biomimetic but non-endogenous lipid bilayer membrane environment may modify the enzyme conformation, while confinement of the large hydrophilic domain with the nanoscale water channels of a continuous lipid bilayer structure may prevent full function of this enzyme. Herein we show that NmEptA remains active despite encapsulation within a nanostructured bicontinuous cubic phase. Full transfer of the phosphoethanolamine (PEA) group from a 1,2-dioleoyl-glycero-phosphoethanolamine (DOPE) doped lipid to monoolein (MO), which makes up the bicontinuous cubic phase, is shown. The reaction was found to be non-specific to the alkyl chain identity. The observed rate of enzyme activity is similar to other membrane bound enzymes, with complete transfer of the PEA group occurring in vitro, under the conditions studied, over a 24 hour timescale.

Detergents in Membrane Protein Purification and Crystallisation.

Detergents play a significant role in structural and functional characterisation of integral membrane proteins (IMPs). IMPs reside in the biological membranes and exhibit a great variation in their structural and physical properties. For in vitro biophysical studies, structural and functional analyses, IMPs need to be extracted from the membrane lipid bilayer environment in which they are found and purified to homogeneity while maintaining a folded and functionally active state. Detergents are capable of successfully solubilising and extracting the IMPs from the membrane bilayers. A number of detergents with varying structure and physicochemical properties are commercially available and can be applied for this purpose. Nevertheless, it is important to choose a detergent that is not only able to extract the membrane protein but also provide an optimal environment while retaining the correct structural and physical properties of the protein molecule. Choosing the best detergent for this task can be made possible by understanding the physical and chemical properties of the different detergents and their interaction with the IMPs. In addition, understanding the mechanism of membrane solubilisation and protein extraction along with crystallisation requirements, if crystallographic studies are going to be undertaken, can help in choosing the best detergent for the purpose. This chapter aims to present the fundamental properties of detergents and highlight information relevant to IMP crystallisation. The first section of the chapter reviews the physicochemical properties of detergents and parameters essential for predicting their behaviour in solution. The second section covers the interaction of detergents with the biologic membranes and proteins followed by their role in membrane protein crystallisation. The last section will briefly cover the types of detergent and their properties focusing on custom designed detergents for membrane protein studies.

Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis.

Examination of protein structure at the subatomic level is required to improve the understanding of enzymatic function. For this purpose, X-ray diffraction data have been collected at 100 K from cholesterol oxidase crystals using synchrotron radiation to an optical resolution of 0.94 Å. After refinement using the spherical atom model, nonmodelled bonding peaks were detected in the Fourier residual electron density on some of the individual bonds. Well defined bond density was observed in the peptide plane after averaging maps on the residues with the lowest thermal motion. The multipolar electron density of the protein-cofactor complex was modelled by transfer of the ELMAM2 charge-density database, and the topology of the intermolecular interactions between the protein and the flavin adenine dinucleotide (FAD) cofactor was subsequently investigated. Taking advantage of the high resolution of the structure, the stereochemistry of main-chain bond lengths and of C=O···H-N hydrogen bonds was analyzed with respect to the different secondary-structure elements.

Production and characterization of recombinant perdeuterated cholesterol oxidase.

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.

Binding and channeling of alternative substrates in the enzyme DmpFG: a molecular dynamics study.

DmpFG is a bifunctional enzyme comprised of an aldolase subunit, DmpG, and a dehydrogenase subunit, DmpF. The aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. In this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, DmpG, using molecular dynamics. All substrates investigated are easily accommodated within the active site, binding with free energy values comparable to the physiological substrate 4-hydroxy-2-ketovalerate. Subsequently, umbrella sampling was utilized to obtain free energy surfaces for the aldehyde intermediates (which would be generated from the aldolase reaction on each of these substrates) to move through the channel to the dehydrogenase DmpF. Small substrates were channeled with limited barriers in an energetically feasible process. We show that the barriers preventing bulky intermediates such as benzaldehyde from moving through the wild-type protein can be removed by selective mutation of channel-lining residues, demonstrating the potential for tailoring this enzyme to allow its use for the synthesis of specific chemical products. Furthermore, positions of transient escape routes in this flexible channel were determined.

High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization.

Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Šresolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Šresolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features.

Exploring ligand recognition, selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana.

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C-termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C-terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C-termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57ßS/H70ßS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a ß-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1ß), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

Biological channeling of a reactive intermediate in the bifunctional enzyme DmpFG.

It has been hypothesized that the bifunctional enzyme DmpFG channels its intermediate, acetaldehyde, from one active site to the next using a buried intermolecular channel identified in the crystal structure. This channel appears to switch between an open and a closed conformation depending on whether the coenzyme NAD(+) is present or absent. Here, we applied molecular dynamics and metadynamics to investigate channeling within DmpFG in both the presence and absence of NAD(+). We found that substrate channeling within this enzyme is energetically feasible in the presence of NAD(+) but was less likely in its absence. Tyr-291, a proposed control point at the channel's entry, does not appear to function as a molecular gate. Instead, it is thought to orientate the substrate 4-hydroxy-2-ketovalerate in DmpG before reaction occurs, and may function as a proton shuttle for the DmpG reaction. Three hydrophobic residues at the channel's exit appear to have an important role in controlling the entry of acetaldehyde into the DmpF active site.