Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.     

Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution

Analysis of the three-dimensional structures of two closely related thermophilic and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Entamoeba histolytica (EhADH1) and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro275) at the center of the dimerization interface might be crucial for maintaining the thermal stability of TbADH. To assess the contribution of Pro275 to the thermal stability of the ADHs, we applied site-directed mutagenesis to replace Asp275 of EhADH1 with Pro (D275P-EhADH1) and conversely Pro275 of TbADH with Asp (P275D-TbADH). The results indicate that replacing Asp275 with Pro significantly enhances the thermal stability of EhADH1 (DeltaT(1/2) 相似文献   

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior

Cofactor specificity in the aldo‐keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site‐directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild‐type. Results of previous pre‐steady‐state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2′‐phosphate of the cofactor. Pre‐steady‐state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2′‐phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. Biotechnol. Bioeng. 2010;107: 763–774. © 2010 Wiley Periodicals, Inc.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution

A new phytase (APPA) with optimum pH 2.5—substantially lower than that of most of microbial phytases (pH 4.5–6.0)—was cloned from Yersinia frederiksenii and heterologously expressed in Escherichia coli. Containing the highly conserved motifs typical of histidine acid phosphatases, APPA has the highest identity (84%) to the Yersinia intermedia phytase (optimal pH 4.5), a member of histidine acid phosphatase family. Based on sequence alignment and molecular modeling of APPA and related phytases, APPA has only one divergent residue, Ser51, in close proximity to the catalytic site. To understand the acidic adaptation of APPA, five mutants (S51A, S51T, S51D, S51K, and S51I) were constructed by site‐directed mutagenesis, expressed in E. coli, purified, and characterized. Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively, confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics—presumably the side‐chain structure near the active site—contributes to the optimal pH for APPA activity. Compared with wild‐type APPA, mutant S51T showed higher specific activity, greater activity over pH 2.0–5.5, and increased thermal and acid stability. These properties make S51T a better candidate than the wild‐type APPA for use in animal feed. Biotechnol. Bioeng. 2009;103: 857–864. © 2009 Wiley Periodicals, Inc.     

The reduced stability of a plant alcohol dehydrogenase is due to the substitution of serine for a highly conserved phenylalanine residue

The zinc-binding long-chain alcohol dehydrogenases from plants and animals exhibit a considerable level of amino acid sequence conservation. While the functional importance of many of the conserved residues is known, the role of others has not yet been determined. We have identified a naturally occurring Adh-1 allele in the legume Phaseolus acutifolius with several unusual characteristics. Individuals homozygous for this allele, Adh-1CN, possess a single isozyme starch gel electrophoretic pattern suggestive of a null allele, and exhibit ADH enzyme activity levels ca. 60% lower than the standard wild-type Adh-1F line. Interestingly, analysis of Adh-1CN homozygotes on an alternative gel system indicates that Adh-1CN does encode a polypeptide capable of forming functional homo- and heterodimers. However, the levels of ADH activity displayed by these isozymes are far lower than those observed for the corresponding wild type ADH-1F isozymes. Dialysis experiments indicate that isozymes containing the ADH-1CN polypeptide are inactivated by slightly acidic conditions, which may explain the apparent null phenotype on starch gels. Elevated temperatures cause a similar loss of enzyme activity. The deduced amino acid sequences of ADH-1CN and ADH-1F were obtained from their corresponding cDNA clones, and the only significant difference detected between the two is a single amino acid replacement substitution. Residue 144 is occupied by phenylalanine in the ADH-1F polypeptide, whereas serine occupies this position in the ADH-1CN polypeptide. The proximity of residue 144 to the catalytic zinc in the substrate-binding pocket, coupled with the fact that it is integral to a defined hydrophobic core of the ADH polypeptide, may explain the observed disruptive effect that the serine substitution has on both the activity and stability of the ADH-1CN polypeptide. It also provides an explanation for the maintenance of phenylalanine or the structurally similar tyrosine at this residue in Zn-binding long-chain ADHs.     

Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.     

Structures of dimeric dihydrodiol dehydrogenase apoenzyme and inhibitor complex: probing the subunit interface with site-directed mutagenesis

Dimeric dihydrodiol dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 A resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two betaalphabetaalphabeta motifs at the N-terminus and an eight-stranded, predominantly antiparallel beta-sheet at the C-terminus. The active-site of DD, occupied either by a glycerol molecule or the inhibitor 4-hydroxyacetophenone, is located in the C-terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the beta-sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site-directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose-fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene.     

Crystal structure of quinone‐dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates

The quinone‐dependent alcohol dehydrogenase (PQQ‐ADH, E.C. 1.1.5.2) from the Gram‐negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three‐dimensional (3D) structures of the native form, with PQQ and a Ca²⁺ ion, and of the enzyme in complex with a Zn²⁺ ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ‐ADH displays an eight‐bladed β‐propeller fold, characteristic of Type I quinone‐dependent methanol dehydrogenases. However, three of the four ligands of the Ca²⁺ ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ‐ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ‐dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.     

Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

Side‐chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L‐leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4–6% of capacity) while remaining remarkably responsive to ADP/L‐leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L‐leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain (‘antenna’), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg‐1 change essentially replicated the Arg443Ser‐1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443‐1 or Arg409‐1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser‐1 mutant protein, obtained in recombinant form, regained most of the wild‐type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg‐2 mutant acquired most of the hGDH1 properties. Hence, side‐chain interactions between 409 and 443 positions in the ‘antenna’ region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.

     

Mapping of the active site of alcohol dehydrogenase with low-molecular ligands

In search of an active alcohol dehydrogenase inhibitor, the structure of which may serve as the basis for a potential drug design, the active site of alcohol dehydrogenase containing NAD and Zn²⁺ ions was mapped using the method of molecular mechanics. Molecular docking was performed using a number of ligands containing characteristic functional groups: formate ion, ammonia, ammonium ion, methanol, and methylamine. Sites of preferable binding were revealed for each ligand and arranged in order of decreasing energy of binding to the enzyme. A comparison of the predicted ligand-binding sites and the experimental data on the location of water and inhibitor binding sites in the known structures of corresponding alcohol dehydrogenase complexes indicated a coincidence of the complex formation sites, which confirms the validity of the method and provides the requirements for a highly effective inhibitor (the pharmacophore model).     

Glu742 substitution to Lys enhances the EDTA tolerance ofEscherichia coli PQQ glucose dehydrogenase

Summary Based on homology analysis of the PQQ (pyrroloquinoline quinone) glucose dehydrogenase (PQQGDH) gene fromEscherichia coli andAcinetobacter calcoaceticus, Glu742 was substituted to Lys by site directed mutagenesis of theE. coli PQQGDH gene (gcd). The mutant enzyme, E742K showed higher tolerance towards EDTA inactivation than wild type PQQGDH. This is the first mutagenesis study of putative a PQQ binding site in PQQ enzyme.     

Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.     

Inhibition of nicotinoprotein (NAD+-containing) alcohol dehydrogenase by trans-4-(N,N-dimethylamino)-cinnamaldehyde binding to the active site

Ethanol oxidation by nicotinoprotein alcohol dehydrogenase (np-ADH) from the bacterium Amycolatopsis methanolica is inhibited by trans-4-(N,N-dimethylamino)-cinnamaldehyde through direct binding to the catalytic zinc ion in a substrate-like geometry. This binding is accompanied by a characteristic red shift of the aldehyde absorbance from 398 nm to 467 nm. Np-ADH is structurally related to mammalian ADH class I, and a model of np-ADH shows how the cinnamaldehyde derivative can be accommodated in the active site of the nicotinoprotein, correlating the structural and enzymological data.     

Crystal structures of ligand‐bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain‐length determining mechanisms

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C₄₀ long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc.     

A single amino acid in the second transmembrane domain of GABA ρ subunits is a determinant of the response kinetics of GABAC receptors

The ρ subunits that constitute the γ‐aminobutyric acid (GABA)_C receptors of retinal neurons form a unique subclass of ligand‐gated chloride channels that give rise to sustained GABA‐evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA ρ1B and ρ2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site‐directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABA_C receptors. When the site contains a proline residue, as in wild‐type ρ1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild‐type ρ2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA ρ receptors, e.g., when the serine residue of the ρ2A receptor was changed to proline, the response of the mutant receptor to imidazole‐4‐acetic acid (I4AA) mimicked that of the ρ1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various ρ receptor subunits. It is noteworthy that the proline residue in native ρ1 subunits and the serine residue of ρ2 subunits are well conserved in all species, a good indication that the presence of multiple GABA ρ subunits serves to generate GABA_C receptors that display the wide range of response kinetics observed on various types of retinal neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 67–76, 1999     

Tolerance to the substitution of buried apolar residues by charged residues in the homologous protein structures

Occurrence and accommodation of charged amino acid residues in proteins that are structurally equivalent to buried non-polar residues in homologues have been investigated. Using a dataset of 1,852 homologous pairs of crystal structures of proteins available at 2A or better resolution, 14,024 examples of apolar residues in the structurally conserved regions replaced by charged residues in homologues have been identified. Out of 2,530 cases of buried apolar residues, 1,677 of the equivalent charged residues in homologues are exposed and the rest of the charged residues are buried. These drastic substitutions are most often observed in homologous protein pairs with low sequence identity (<30%) and in large protein domains (>300 residues). Such buried charged residues in the large proteins are often located in the interface of sub-domains or in the interface of structural repeats, Beyond 7A of residue depth of buried apolar residues, or less than 4% of solvent accessibility, almost all the substituting charged residues are buried. It is also observed that acidic sidechains have higher preference to get buried than the positively charged residues. There is a preference for buried charged residues to get accommodated in the interior by forming hydrogen bonds with another sidechain than the main chain. The sidechains interacting with a buried charged residue are most often located in the structurally conserved regions of the alignment. About 50% of the observations involving hydrogen bond between buried charged sidechain and another sidechain correspond to salt bridges. Among the buried charged residues interacting with the main chain, positively charged sidechains form hydrogen bonds commonly with main chain carbonyls while the negatively charged residues are accommodated by hydrogen bonding with the main chain amides. These carbonyls and amides are usually located in the loops that are structurally variable among homologous proteins.