A template RNA entry channel in the fingers domain of the poliovirus polymerase

Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase, 3D(pol), that is required for viral RNA replication. Structures of 3D(pol) from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a series of alanine substitution mutations at these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex (EC) stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding, while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for EC stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3D(pol) pinky finger drastically decreased the rate of initiation as well as EC stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or EC stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA ECs.     

Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA

The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k(cat), while more noticeable affects were observed in K(m). The mutant D370A/Y540F showed the most significant changes in the pH dependence of k(cat)/K(m) and K(m) relative to wild-type PutA86-601 with an apparent pK(a) value of about 8.2 for the pH-dependent decrease in K(m). From the pH profile of D370A/Y540F inhibition by l-tetrahydro-2-furoic acid (l-THFA), the pH dependency of K(m) in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the E(m) for bound FAD. At pH 7.0, E(m) values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in E(m) determined for D370A relative to wild-type PutA86-601 (E(m)=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.     

Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3

The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme's NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron.     

Identification of the active site of human mitochondrial malonyl‐coenzyme a decarboxylase: A combined computational study

Malonyl‐CoA decarboxylase (MCD) can control the level of malonyl‐CoA in cell through the decarboxylation of malonyl‐CoA to acetyl‐CoA, and plays an essential role in regulating fatty acid metabolism, thus it is a potential target for drug discovery. However, the interactions of MCD with CoA derivatives are not well understood owing to unavailable crystal structure with a complete occupancy in the active site. To identify the active site of MCD, molecular docking and molecular dynamics simulations were performed to explore the interactions of human mitochondrial MCD (HmMCD) and CoA derivatives. The findings reveal that the active site of HmMCD indeed resides in the prominent groove which resembles that of CurA. However, the binding modes are slightly different from the one observed in CurA due to the occupancy of the side chain of Lys183 from the N‐terminal helical domain instead of the adenine ring of CoA. The residues 300 ? 305 play an essential role in maintaining the stability of complex mainly through hydrogen bond interactions with the pyrophosphate moiety of acetyl‐CoA. Principle component analysis elucidates the conformational distribution and dominant concerted motions of HmMCD. MM_PBSA calculations present the crucial residues and the major driving force responsible for the binding of acetyl‐CoA. These results provide useful information for understanding the interactions of HmMCD with CoA derivatives. Proteins 2016; 84:792–802. © 2016 Wiley Periodicals, Inc.     

Crystal structure analysis,covalent docking,and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3‐hydroxybutyrate) depolymerase were identified in two high‐resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281–295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3‐hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281–295 in comparison to the apo (substrate‐free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281–295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351–1361. © 2017 Wiley Periodicals, Inc.     

Insights into the role of d-amino acid oxidase mutations in amyotrophic lateral sclerosis

Missense mutations in the coding region of d -amino acid oxidase (DAO) have been found in patients suffering from amyotrophic lateral sclerosis (ALS). Mutations primarily impair the enzymatic activity of DAO and cause neurodegeneration due to an abnormal accumulation of d -serine in the spinal cord. However, the structural and dynamic changes that lead to impaired enzymatic activity are not fully understood. We present here extensive molecular dynamics simulations of wild-type, and all reported ALS-associated DAO mutants to elucidate the plausible mechanisms of impaired enzymatic activity, a critical function needed for neuroprotection. Simulation results show that DAO mutations disrupt several key interactions with the active site residues and decrease the conformational flexibility of active site loop comprising 216 to 228 residues, necessary for substrate binding and product release. This conformational restriction of the active site loop in the mutants is mainly due to the distortion of critical salt bridge and hydrogen bond interactions compared with wild-type. Furthermore, binding free energy calculations show that DAO mutants have a lower binding affinity toward cofactor flavin adenine dinucleotide and substrate imino-serine than the wild-type. A closer look at the cofactor and substrate interaction profiles further show that DAO mutants have lost several critical interactions with the neighboring residues as compared with wild-type. Taken together, this study provides first-hand explanation of crucial structural features that lead to the loss of enzymatic function in DAO mutants and highlights the need of further genomic scans of patients with ALS to map the association of novel DAO variants in ALS pathophysiology.     

Differential contributions of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis

Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg(454), Tyr(456), Cys(472), Gly(488), Thr(491), and Trp(677)) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr(456), which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4'-hydroxyl, or Arg(454), which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity. In contrast, substitution of Thr(491), which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456S K(m)(cyt )(c) and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp(677) is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp(677) and the carboxyl-terminal Ser(678) decreased catalytic activity 50-fold without affecting FAD content.     

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium‐bound and calcium‐free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure‐based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Biochemical analysis and structure determination of Paucimonas lemoignei poly(3‐hydroxybutyrate) (PHB) depolymerase PhaZ7 muteins reveal the PHB binding site and details of substrate–enzyme interactions

Five amino acids (Y105, Y176, Y189, Y189, W207) that constitute the substrate binding site of PHB depolymerase PhaZ7 were identified. All residues are located at a single surface‐exposed location of PhaZ7. Exchange of these amino acids by less hydrophobic, hydrophilic or negatively charged residues reduced binding of PhaZ7 to PHB. Modifications of other residues at the PhaZ7 surface (F9, Y66, Y103, Y124, Y169, Y172, Y173, F198, Y203, Y204, F251, W252) had no effect on substrate binding. The PhaZ7 wild‐type protein, three muteins with single amino acid exchanges (Y105A, Y105E, Y190E), a PhaZ7 variant with deletion of residues 202–208, and PhaZ7 in which the active‐site serine had been replaced by alanine (S136A) were crystallized and their structures were determined at 1.6–2.0 Å resolution. The structures were almost identical but revealed flexibility of some regions. Structural analysis of PhaZ7 (S136A) with bound 3‐hydroxybutyrate tetramer showed that the substrate binds in a cleft that is composed of Y105, Y176, Y189 and Y190 and thus confirmed the data obtained by site‐directed mutagenesis. To the best of our knowledge this is the first example in which the substrate binding site of a PHB depolymerase is documented at a molecular and structural level.     

Mechanism of FAD reduction and role of active site residues His-225 and Tyr-259 in Arthrobacter globiformis dimethylglycine oxidase: analysis of mutant structure and catalytic function

Residues His-225 and Tyr-259 are located close to the FAD in the dehydrogenase active site of the bifunctional dimethylglycine oxidase (DMGO) of Arthrobacter globiformis. We have suggested [Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038-4048] that these residues are involved in abstraction of a proton from the substrate amine group of dimethylglycine prior to C-H bond breakage and FAD reduction. To investigate this proposal, we have isolated two mutant forms of DMGO in which (i) His-225 is replaced with Gln-225 (H225Q mutant) and (ii) Tyr-259 is replaced with Phe-259 (Y259F mutant). Both mutant enzymes retain the ability to oxidize substrate, but the steady-state turnover of the Y259F mutant is attenuated more than 200-fold. Only modest changes in kinetic parameters are observed for the H225Q mutant during steady-state turnover. Stopped-flow studies indicate that the rate of FAD reduction in the Y259F enzyme is substantially impaired by a factor of approximately 1500 compared with that of the wild-type enzyme, suggesting a key role for this residue in the reductive half-reaction of the enzyme. The kinetics of FAD reduction in the H225Q enzyme are complex and involve three discrete kinetic phases that are attributed to different conformational states of this mutant, evidence for which is provided by crystallographic analysis. Neither the H225Q enzyme nor the Y259F enzyme stabilizes the FADH(2)-iminium charge-transfer complex observed previously in stopped-flow studies with the wild-type enzyme. Our studies are consistent with a key role for Tyr-259, but not His-225, in deprotonation of the substrate amine group prior to FAD reduction. We infer that residue His-225 is likely to modulate the acid-base properties of Tyr-259 by perturbing the pK(a) of Tyr-259 and thus fine-tunes the reaction chemistry to facilitate proton abstraction under physiological conditions. Our data are discussed in the context of the crystallographic data for DMGO and also in relation to contemporary mechanisms for flavoprotein-catalyzed oxidation of amine substrates.     

Crystal structure of UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) from Mycobacterium tuberculosis

The biosynthesis of UDP-N-acetylmuramic acid (UDP-MurNAc) by reduction of UDP-N-acetylglucosamine-enolpyruvate (UDP-GlcNAc-EP) in an NADPH and FAD-dependent reaction in bacteria is one of the key steps in peptidoglycan biosynthesis catalyzed by UDP-N-acetylglucosamine-enolpyruvate reductase (MurB). Here, we present the crystal structure of Mycobacterium tuberculosis MurB (MtbMurB) with FAD as the prosthetic group at 2.0 Å resolution. There are six molecules in asymmetric unit in the form of dimers. Each protomer can be subdivided into three domains and the prosthetic group, FAD is bound in the active site between domain I and domain II. Comparison of MtbMurB structure with the structures of the Escherichia coli MurB (in complex with UDP-GlcNAc-EP) and Pseudomonas aeruginosa MurB (in complex with NADPH) showed all three structures share similar domain architecture and residues in the active site. The nicotinamide and the enol pyruvyl moieties are well aligned upon superimposition, both positioned in suitable position for hydride transfer to and from FAD. The comparison studies and MD simulations demonstrate that the two lobes of domain-III become more flexible. The substrates (NADPH and UDP-GlcNAc-EP) binding responsible for open conformation of MurB, suggesting that NADPH and UDP-GlcNAc-EP interactions are conformationally stable. Our findings provide a detail mechanism about the closed to open state by binding of NADPH and UDP-GlcNAc-EP induces the conformational changes of MurB structure that may trigger the MurB catalytic reaction.     

Exploration micromechanism of VP35 IID interaction and recognition dsRNA: A molecular dynamics simulation

Multifunctional viral protein (VP35) encoded by the highly pathogenic Ebola viruses (EBOVs) can antagonize host double‐stranded RNA (dsRNA) sensors and immune response because of the simultaneous recognition of dsRNA backbone and blunt ends. Mutation of select hydrophobic conserved basic residues within the VP35 inhibitory domain (IID) abrogates its dsRNA‐binding activity, and impairs VP35‐mediated interferon (IFN) antagonism. Herein the detailed binding mechanism between dsRNA and WT, single mutant, and double mutant were investigated by all‐atom molecular dynamics (MD) simulation and binding energy calculation. R312A/R322A double mutations results in a completely different binding site and orientation upon the structure analyses. The calculated binding free energy results reveal that R312A, R322A, and K339A single mutations decrease the binding free energies by 17.82, 13.18, and 13.68 kcal mol^?1, respectively. The binding energy decomposition indicates that the strong binding affinity of the key residues is mainly due to the contributions of electrostatic interactions in the gas phase, where come from the positively charged side chain and the negatively charged dsRNA backbone. R312A, R322A, and K339A single mutations have no significant effect on VP35 IID conformation, but the mutations influence the contributions of electrostatic interactions in the gas phase. The calculated results reveal that end‐cap residues which mainly contribute VDW interactions can recognize and capture dsRNA blunt ends, and the central basic residues (R312, R322, and K339) which mainly contribute favorable electrostatic interactions with dsRNA backbone can fix dsRNA binding site and orientation. Proteins 2017; 85:1008–1023. © 2017 Wiley Periodicals, Inc.     

Gain‐of‐function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor,Aer

The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD‐containing PAS domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS domain and found 72 aerotaxis‐defective mutants, 24 of which were gain‐of‐function, signal‐on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS–FAD domain in NifL from Azotobacter vinlandii. Signal‐on lesions clustered in the FAD binding pocket, the β‐scaffolding and in the N‐cap loop. We suggest that the signal‐on lesions mimic the ‘signal‐on’ state of the PAS domain, and therefore may be markers for the signal‐in and signal‐out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the β‐scaffolding and the N‐cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS β‐scaffold and residue Q248C in the HAMP AS‐2 helix.     

Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase

Acyl-CoA oxidase (ACO) catalyzes the first and rate-determining step of the peroxisomal beta-oxidation of fatty acids. The crystal structure of ACO-II, which is one of two forms of rat liver ACO (ACO-I and ACO-II), has been solved and refined to an R-factor of 20.6% at 2.2-A resolution. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. The X-ray analysis showed that the overall folding of ACO-II less C-terminal 221 residues is similar to that of medium-chain acyl-CoA dehydrogenase (MCAD). However, the N-terminal alpha- and beta-domains rotate by 13 with respect to the C-terminal alpha-domain compared with those in MCAD to give a long and large crevice that accommodates the cofactor FAD and the substrate acyl-CoA. FAD is bound to the crevice between the beta- and C-terminal domains with its adenosine diphosphate portion interacting extensively with the other subunit of the molecule. The flavin ring of FAD resides at the active site with its si-face attached to the beta-domain, and is surrounded by active-site residues in a mode similar to that found in MCAD. However, the residues have weak interactions with the flavin ring due to the loss of some of the important hydrogen bonds with the flavin ring found in MCAD. The catalytic residue Glu421 in the C-terminal alpha-domain seems to be too far away from the flavin ring to abstract the alpha-proton of the substrate acyl-CoA, suggesting that the C-terminal domain moves to close the active site upon substrate binding. The pyrimidine moiety of flavin is exposed to the solvent and can readily be attacked by molecular oxygen, while that in MCAD is protected from the solvent. The crevice for binding the fatty acyl chain is 28 A long and 6 A wide, large enough to accommodate the C23 acyl chain.     

In silico analyses of substrate interactions with human serum paraoxonase 1

Human paraoxonase (HuPON1) is a serum enzyme that exhibits a broad spectrum of hydrolytic activities, including the hydrolysis of various organophosphates, esters, and recently identified lactone substrates. Despite intensive site-directed mutagenesis and other biological studies, the structural basis for the specificity of substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The results suggest that the active site of HuPON1 is characterized by two distinct binding regions: the hydrophobic binding site for arylesters/lactones, and the paraoxon binding site for phosphotriesters. The unique binding modes proposed for each type of substrate reveal a number of key residues governing substrate specificity. The polymorphic residue R/Q192 interacts with the leaving group of paraoxon, suggesting it plays an important role in the proper positioning of this substrate in the active site. MD simulations of the optimal binding complexes show that residue Y71 undergoes an "open-closed" conformational change upon ligand binding, and forms strong interactions with substrates. Further binding free energy calculations and residual decomposition give a more refined molecular view of the energetics and origin of HuPON1/substrate interactions. These studies provide a theoretical model of substrate binding and specificity associated with wild type and mutant forms of HuPON1, which can be applied in the rational design of HuPON1 variants as bioscavengers with enhanced catalytic activity.     

Molecular recognition of Fc‐specific ligands binding onto the consensus binding site of IgG: insights from molecular simulation

Immunoglobulin G (IgG) plays an important role in clinical diagnosis and therapeutics. Meanwhile, the consensus binding site (CBS) on the Fc domain of IgG is responsible for ligand recognition, especially for Fc‐specific ligands. In this study, molecular simulation methods were used to investigate molecular interactions between the CBS of the Fc domain and seven natural Fc‐specific ligands. The analysis on the binding energy of the Fc–ligand complex indicated that hydrophobic interactions provide the main driving force for the Fc–ligand binding processes. The hot spots on the ligands and Fc were identified with the computational alanine scanning approach. It was found that the residues of tryptophan and tyrosine on the ligands have significant contributions for the Fc–ligand binding, while Met252, Ile253, Asn434, His435, and Tyr436 are the key residues of Fc. Moreover, two binding modes based on tryptophan or tyrosine were summarized and constructed according to the pairwise interaction analysis. Guidelines for the rational design of CBS‐specific ligands with high affinity and specificity were proposed. Copyright © 2014 John Wiley & Sons, Ltd.     

Crystallographic analysis of active site contributions to regiospecificity in the diiron enzyme toluene 4-monooxygenase

Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric μ-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH(2)-benzoate and p-Br-benzoate showed a μ-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a π-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 ?) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release.     

Molecular modeling of formate dehydrogenase: the formation of the Michaelis complex

The formation of the reactive enzyme–substrate complex of formate dehydrogenase has been investigated by molecular dynamics techniques accounting for different conformational states of the enzyme. Simulations revealed that the transport of substrate to the active site through the substrate channel proceeds in the open conformation of enzyme due to the crucial role of the Arg284 residue acting as a vehicle. However, formate binding in the active site of the open conformation leads to the formation of a nonproductive enzyme–substrate complex. The productive Michaelis complex is formed only in the closed enzyme conformation after the substrate and coenzyme have bound, when required rigidity of the binding site and reactive formate orientation due to interactions with Arg284, Asn146, Ile122, and His332 residues is attained. Then, the high occupancy (up to 75%) of the reactive substrate–coenzyme conformation is reached, which was demonstrated by hybrid quantum mechanics/molecular mechanics simulations using various semiempirical Hamiltonians.     

Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases

Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb3 oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb3 oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm.     

15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S(2)) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S(2) values, whereas the S(2) values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.