Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

A Remote Palm Domain Residue of RB69 DNA Polymerase Is Critical for Enzyme Activity and Influences the Conformation of the Active Site

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol^D714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k_pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.     

Structure of the Homodimeric Glycine Decarboxylase P-protein from Synechocystis sp. PCC 6803 Suggests a Mechanism for Redox Regulation

Glycine decarboxylase, or P-protein, is a pyridoxal 5′-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α₂ homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys⁹⁷² in the C terminus and Cys³⁵³ located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys⁹⁷² and Cys³⁵³ by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353–972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.     

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn²⁺-dependent dipeptidase. The crystal structure of this protein in the Mn²⁺-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys¹⁵⁵ and Cys¹⁷⁸, are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.     

Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 Å resolution

The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 Å resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 Å resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R-value of 37.9%.There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic.The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369.No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.     

Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport

Na⁺/H⁺ antiporters have a crucial role in pH and Na⁺ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133.     

Crystal structure of (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha

(R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha H16 (RePhaB) is an enzyme that catalyzes the NADPH-dependent reduction of acetoacetyl-CoA, an intermediate of polyhydroxyalkanoates (PHA) synthetic pathways. Polymeric PHA is used to make bioplastics, implant biomaterials, and biofuels. Here, we report the crystal structures of RePhaB apoenzyme and in complex with either NADP⁺ or acetoacetyl-CoA, which provide the catalytic mechanism of the protein. RePhaB contains a Rossmann fold and a Clamp domain for binding of NADP⁺ and acetoacetyl-CoA, respectively. The NADP⁺-bound form of RePhaB structure reveals that the protein has a unique cofactor binding mode. Interestingly, in the RePhaB structure in complex with acetoacetyl-CoA, the conformation of the Clamp domain, especially the Clamp-lid, undergoes a large structural change about 4.6 Å leading to formation of the substrate pocket. These structural observations, along with the biochemical experiments, suggest that movement of the Clamp-lid enables the substrate binding and ensures the acetoacetyl moiety is located near to the nicotinamide ring of NADP⁺.     

Biochemical and structural characterisation of dehydroquinate synthase from the New Zealand kiwifruit Actinidia chinensis

One of the novel aspects of kiwifruit is the presence of a high level of quinic acid which contributes to the flavour of the fruit. Quinic acid metabolism intersects with the shikimate pathway, which is responsible for the de novo biosynthesis of primary and secondary aromatic metabolites. The gene encoding the enzyme which catalyses the second step of the shikimate pathway, dehydroquinate synthase (DHQS), from the New Zealand kiwifruit Actinidia chinensis was identified, cloned and expressed. A. chinensis DHQS was activated by divalent metal ions, and was found to require NAD⁺ for catalysis. The protein was crystallised and the structure was solved, revealing a homodimeric protein. Each monomer has a NAD⁺ binding site nestled between the distinct N- and C-terminal domains. In contrast to other microbial DHQSs, which show an open conformation in the absence of active site ligands, A. chinensis DHQS adopts a closed conformation. This is the first report of the structure of a DHQS from a plant source.     

Substrate positions and induced-fit in crystalline adenylate kinase.

The binding positions of ATP and AMP in pig muscle adenylate kinase (EC 2.7.4.3) have been located by X-ray diffraction analysis. For this purpose crystals have been soaked with solutions containing substrates and substrate analogues. Two adenosine pockets and the region of the phosphates have been identified. In combination with other experimental data the pockets have been assigned to the AMP site and the ATP site, respectively. Moreover, the results suggest that the known conformations of adenylate kinase reflect an induced-fit of the enzyme: conformation B being related to the free enzyme E and conformation A being related to E¹, the enzyme species after a substrate-induced conformational change.     

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes

Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M₄ lactate dehydrogenase structures. One of these is the 2.0 Å resolution apoenzyme structure; the other is a 3.0 Å resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase²: NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.     

E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb⁺ deocclusion stimulated by Na⁺ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb⁺ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb⁺ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb⁺]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb⁺-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na⁺-stimulated Rb⁺ deocclusion would require the release of at least one Rb⁺ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb⁺ in equilibrium conditions. Results show that, while Mg^2 + decreases the affinity for Rb⁺, addition of vanadate offsets this effect, increasing the affinity for Rb⁺. In transient experiments, we investigated the exchange of Rb⁺ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na⁺ without significantly affecting the rate of Rb⁺ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb⁺ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb⁺.     

Analysis of the Conformational Stability and Activity of Candida antarctica Lipase B in Organic Solvents: INSIGHT FROM MOLECULAR DYNAMICS AND QUANTUM MECHANICS/SIMULATIONS*

The conformational stability and activity of Candida antarctica lipase B (CALB) in the polar and nonpolar organic solvents were investigated by molecular dynamics and quantum mechanics/molecular mechanics simulations. The conformation change of CALB in the polar and nonpolar solvents was examined in two aspects: the overall conformation change of CALB and the conformation change of the active site. The simulation results show that the overall conformation of CALB is stable in the organic solvents. In the nonpolar solvents, the conformation of the active site keeps stable, whereas in the polar solvents, the solvent molecules reach into the active site and interact intensively with the active site. This interaction destroys the hydrogen bonding between Ser¹⁰⁵ and His²²⁴. In the solvents, the activation energy of CALB and that of the active site region were further simulated by quantum mechanics/molecular mechanics simulation. The results indicate that the conformation change in the region of active sites is the main factor that influences the activity of CALB.     

Glutathione-independent Formaldehyde Dehydrogenase from Pseudomons putida: Survey of Functional Groups with Special Regard for Cysteine Residues

The role of cysteine residues for structure and function of formaldehyde dehydrogenase from Pseudomonas putida was analysed by amino acid sequence comparison, homology-based structure modeling, site-directed mutagenesis, and chemical modification. Five out of seven cysteine residues found in the enzyme were concluded to coordinate with an active site zinc (Cys-46) and structural zinc atoms (Cys-97, -100, -103, and -111) from the sequence comparison with other Zn-containing medium-chain alcohol dehydrogenase homologues. The three-dimensional structure model based on the known structure of the horse liver E-type alcohol dehydrogenase (ADH) indicated that Cys-257 is located very far from the active site Zn and NAD⁺ binding region, suggesting that Cys-257 does not participate in the enzyme reaction. The structure also suggested that Cys-166 does not coordinate to active site Zn, but Asp-169 functions as a Zn-ligand, instead.     

Insights into Eukaryotic Primer Synthesis from Structures of the p48 Subunit of Human DNA Primase

DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn^2 + but not Mg^2 + significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn^2 + provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.     

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis

Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp¹²⁰ and Lys⁷⁷ is suggested.     

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp ² to sp ³ conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism.     

A New Type of Signal Peptidase Cleavage Site Identified in an RNA Virus Polyprotein

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein E^rns is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the E^rns membrane anchor. Despite the absence of a hydrophobic region, the site between the C terminus of E^rns and E1, the protein located downstream in the polyprotein, is cleaved by signal peptidase, as demonstrated by mutagenesis and inhibitor studies. Thus, E^rnsE1 is processed at a novel type of signal peptidase cleavage site showing a different membrane topology. Prevention of glycosylation or introduction of mutations into the C-terminal region of E^rns severely impairs processing, presumably by preventing proper membrane interaction or disturbing a conformation critical for the protein to be accepted as a substrate by signal peptidase.