Non-conserved Amino Acid Residues Research Articles

Production of 2′-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain

2′-Fucosyllactose (2′-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2′-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2′-FL from glucose and lactose. Thus, the overproduction of 2′-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.

Enhanced thermostability of formate dehydrogenase via semi-rational design

An NAD+-dependent formate dehydrogenase from Lodderomyces elongisporus (LeFDH) was expressed in Escherichia coli and then purified by nickel affinity chromatography. The analysis of enzymatic properties showed that the specific activity of LeFDH was much high (16.26 U·mg–1), while the thermal stability may be insufficient for industrial application. To further enhance the thermal stability by semi-rational design, sequences alignment, homologous modeling and molecular dynamic simulations were conducted. As a result, seven non-conserved amino acid residues far from the active center were selected for site-directed mutagenesis. Several mutants as well as a binary mutant displayed improved thermal stability and unreduced specific activity. Then temperature and pH stability, kinetic parameters and specific activity, and structural characteristics of wild-type and mutant LeFDH were compared and evaluated. The variant K21P/K22R exhibited an increased half-life by 211% at 60 °C and a 4.5 °C higher Tm than those of wild type, and good stability in alkaline environment. This binary mutant enhances the operational stability of LeFDH in the catalytic reaction and thus improves its potential for application in coenzyme regenerations.

Sequence diversity and catalytic properties of phytases

Phytic acid is an antinutritional factor in cereal feeds, and the use of phytases increases the bioavailability of nutrients bound to this molecule. However, the application of these enzymes depends on their thermal stability and activity at acidic pH. Therefore, in this study we created a database composed of 59 phytase sequences and analyzed the interactions that stabilize their structures in order to understand whether they contribute to the biochemical properties observed. The sequences were aligned and grouped at 30 % similarity, generating 5 clusters, which highlights the high variability among them. A comparative structural analysis of the cluster 3 phytases revealed conserved catalytic domains, as well as eight cysteine residues along the primary sequence, forming disulfide bonds for stabilizing the three-dimensional structure. However, the number of Van der Waals, ionic, and hydrogen interactions, and disulfide bonds was not determinant for the biochemical characteristics presented by these enzymes. The phytase KM873028, from cluster 3, was selected for characterization studies, but its expression in Pichia pastoris generated a protein with properties distinct from those derived from the same sequence expressed in a prokaryotic system. It is likely that the differences observed are associated with the location of the interactions in the structures, non-conserved amino acid residues found around the catalytic site, and post-translational modifications inherent to the expression systems. These possibilities highlight the relevance of strategic choices related to enzyme expression aiming at its production and industrial feasibility.

Engineering non-conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34 for improved thermal stability: an in-silico evaluation

GH11 xylanases are commercially important enzymes for degradation of xylan fibers. We have identified the presence of nine non-conserved and five conserved salt bridges in GH11 xylanase from Bacillus pumilus SSP34. We have designed two sets of mutants viz., (1) substitution mutants in which non-conserved charged amino acid residues have been replaced with appropriate hydrophobic residues based on side chain occupancy and hydrophobicity and (2) deletion mutants where non-conserved charged residues have been deleted. The stability of the mutants has been evaluated in-silico by analyzing the contributions of non-covalent interactions like hydrophobic interaction clusters and salt bridges. The stability of the resultant mutants was evaluated using parameters such as radius of gyration, solvent accessible surface area, root mean square deviation, root mean square fluctuations and protein unfolding measurements using molecular dynamic simulations. The deletion of certain charged residues resulted in mutants having lowered radius of gyration and decreased surface areas. However, RMSD and RMSF measurements indicated lowered stability in comparison to substitution mutants. Of the substitution mutants, the SBM 3 was the most stable mutant as indicated by Rg, SASA, RMSF and simulated protein unfolding measurements. The major contributing factors for improved stability could be strengthening of hydrophobic interactions in the GH11 xylanase from B. pumilus. These in-silico stability measurements of salt bridge mutants may lead to better design of GH11 xylanases for commercial applications. Communicated by Ramaswamy H. Sarma

Identification and engineering on the nonconserved residues of metallo-β-lactamase-type thioesterase to improve the enzymatic activity.

The standalone metallo-β-lactamase-type thioesterase (MβL-TE), belongs to the group V nonreducing polyketide synthase agene cluster, catalyzes the rate-limiting step of product releasing. Our work first investigated on the orthologous MβL-TEs from different origins to determine which nonconserved amino acid residues are important to the hydrolysis efficiency. A series of chimeric MβL-TEs were constructed by fragment swapping and site-directed mutagenesis, in vivo enzymatic assay showed that two nonconserved residues A19 and E75 (numbering in HyTE) were critical to the catalytic performance. Protein structure modeling suggested that these two residues are located in different areas of HyTE. A19 is on the entrance to the active sites, whereas E75 resides in the linker between the two β strands which hold the metal-binding sites. Combining with computational simulations and comparative enzymatic assay, different screening criteria were set up for selecting the variants on the two noncatalytic and nonconserved key residues to improve the catalytic activity. The rational design on A19 and E75 gave five candidates in total, two (A19F and E75Q) of which were thus found significantly improved the enzymatic performance of HyTE. The double-point mutant was constructed to further improve the activity, which was increased by 28.4-fold on product accumulation comparing to the wild-type HyTE. This study provides a novel approach for engineering on nonconserved residues to optimize enzymatic performance.

Identification of Amino Acid Residues in Human IgM Fc Receptor (FcµR) Critical for IgM Binding.

Both non-immune “natural” and antigen-induced “immune” IgM are important for protection against infections and for regulation of immune responses to self-antigens. The roles of its Fc receptor (FcµR) in these IgM effector functions have begun to be explored. In the present study, by taking advantage of the difference in IgM-ligand binding of FcµRs of human (constitutive binding) and mouse (transient binding), we replaced non-conserved amino acid residues of human FcµR with mouse equivalents before establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resultant eight-different mutant FcµR-bearing cells were compared with WT receptor-bearing cells for cell-surface expression and IgM-binding by flow cytometric assessments using receptor-specific mAbs and IgM paraproteins as ligands. Three sites Asn66, Lys79-Arg83, and Asn109, which are likely in the CDR2, DE loop and CDR3 of the human FcµR Ig-like domain, respectively, were responsible for constitutive IgM binding. Intriguingly, substitution of Glu41 and Met42 in the presumed CDR1 with the corresponding mouse residues Gln and Leu, either single or more prominently in combination, enhanced both the receptor expression and IgM binding. A four-aa stretch of Lys24-Gly27 in the predicted A ß-strand of human FcµR appeared to be essential for maintenance of its proper receptor conformation on plasma membranes because of reduction of both receptor expression and IgM-binding potential when these were mutated. Results from a computational structural modeling analysis were consistent with these mutational data and identified a possible mode of binding of FcµR with IgM involving the loops including Asn66, Arg83 and Asn109 of FcµR interacting principally with the Cµ4 domain including Gln510 and to a lesser extent Cµ3 domain including Glu398, of human IgM. To our knowledge, this is the first experimental report describing the identification of amino acid residues of human FcµR critical for binding to IgM Fc.

Drysdalin, a snake neurotoxin with higher affinity for soluble acetylcholine binding protein from Aplysia californica than from Lymnaea stagnalis

Acetylcholine binding proteins (AChBPs), structural and functional surrogates of the extracellular binding domain of nicotinic acetylcholine receptor (nAChRs), in complex with various antagonists and agonists have provided detailed insights into the neurotransmitter binding site of nAChRs. The classical long-chain α-neurotoxins bungarotoxin (44-fold) and cobratoxin (7-fold) bind to Lymnaea stagnalis (Ls)-AChBP with higher affinity compared to Aplysia californica (Ac)-AChBP. In this study, we describe a novel long chain α-neurotoxin Drysdalin, which has higher binding affinity (7-fold) to Ac-AChBP when compared to Ls-AChBP. This suggests an involvement of different regions or modes of interaction of drysdalin, when compared to the bungarotoxin and cobratoxin. We also found that the C-terminal 24-amino acid residues of drysdalin are critical for the binding to Ac-AChBP and its removal caused ~90-fold reduction in affinity. Further to understand the interaction of drysdalin with Ac-AChBP, we studied the role of three non-conserved amino acid residues of drysdalin, namely Arg30, Leu34 and Ala37. Substitution of Arg30 with the conserved Phe residue caused a ~100-fold reduction, Leu34 with conserved Arg caused a ~6-fold reduction, whereas substitution of Ala37 with conserved Arg enhanced the binding by 3-fold. The dramatic influence of this carboxyl terminal sequence enriched in arginine and proline residues suggests that the toxin binding pose is influenced primarily by this extended sequence.

In Silico Finding of Key Interaction Mediated α3β4 and α7 Nicotinic Acetylcholine Receptor Ligand Selectivity of Quinuclidine-Triazole Chemotype.

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes α3β4 and α7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary β4-subunit of the α3β4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the α3β4 selectivity of the quinuclidine-triazole chemotype, explaining the 47–327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the α7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

Structural characterization of ribT from Bacillus subtilis reveals it as a GCN5-related N-acetyltransferase.

In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Å resolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5-related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-conserved amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis.

Key amino acid residue in Melanocortin-1 receptor (melanocyte α-MSH receptor) for ligand selectivity

The melanocortin-1 receptor (MC1R) is a subtype of the melanocortin receptor family and NDP-α-MSH is a non-selective agonist for MC1R. The core sequence of NDP-α-MSH, His-Phe-Arg-Trp, is important for ligand binding and biological activities at the melanocortin receptor subtypes (MCRs). A recent study indicates that Trp9 in NDP-α-MSH plays an important role in ligand selectivity. Deletion of Trp9 in NDP-α−MSH (des-Trp9-NDP-α-MSH) resulted in loss of agonist activity at MC4R, although remains agonist activity at MC1R. The molecular basis for this receptor ligand selectivity is unknown. In this study we examined what region of the MC1R is responsible for des-NDP-α-MSH selectivity. Our results indicate that (1) substitution of TM3 of MC4R with the corresponding region of MC1R switches des-Trp9-NDP-α-MSH from no activity to agonist; (2) des-Trp9-NDP-α-MSH exhibits agonistic activity at the L133M mutation of the MC4R; and (3) substitution of non-conserved amino acid residue M128 in TM3 of MC1R significantly reduced des-Trp9-NDP-α-MSH agonist activity. Our results demonstrate that amino acid residue 128 in TM3 of MC1R, or amino acid residue L133 in TM3 of the MC4R, play crucial roles in ligand des-Trp9-NDP-α-MSH selectivity at MC1R or MC4R.

Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors.

Geobacter sulfurreducens, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c-type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces.IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells.

Expression and characterization of EF-hand I loop mutants of aequorin replaced with other loop sequences of Ca2+-binding proteins: an approach to studying the EF-hand motif of proteins.

The binding properties of Ca(2+) to EF-hand I of aequorin (AQ) were characterized by replacing the loop sequence of EF-hand I (AQ[I]) with other known loop sequences of Ca(2+)-binding proteins, including photoproteins (aequorin, clytin-I, clytin-II and mitrocomin), Renilla luciferin-binding protein (RLBP) and calmodulin (CaM). For evaluation of the binding affinity of Ca(2+) to AQ[I] mutants, the half-decay time of the maximum intensity in the luminescence reaction triggered by Ca(2+) was used as an indicator and 22 kinds of AQ[I] mutants were expressed in Escherichia coli cells. AQ[I] mutants replaced with the EF-hand I and EF-hand III from photoproteins showed sufficient luminescence activity, but it was not shown by other EF-hands from RLBP and CaM. An AQ[I] mutant with a lysine or arginine residue at the second position of the non-conserved amino acid residue showed a slow-decay pattern of luminescence, indicating that the Ca(2+)-binding affinity to aequorin was reduced by a positive charge at the second position of the loop sequence. The specific loop sequence of the EF-hand I motif in aequorin caused the specific Ca(2+)-triggered luminescence pattern.

Frequency and Epitope Specificity of Anti-Factor VIII C1 Domain Antibodies in Acquired and Hereditary Hemophilia Inhibitor Patient Plasma

Acquired hemophilia A (AHA) is a rare autoimmune disease caused by development of inhibitory anti-factor VIII (fVIII) antibodies (also called inhibitors) resulting in severe hemorrhages. In addition, inhibitor development is the most serious complication of today's replacement therapy in patients with hereditary X-linked hemophilia A (HA) disorder. Earlier studies showed that antibodies in AHA and HA inhibitor plasmas are both primarily directed to the A2 and C2 domains suggesting that these two domains are the predominant immunogenic fVIII regions (Fulcher et al, 1987; Prescott et al, 1997; Lollar, 2004). However, the C1 domain also makes a major contribution to the humoral anti-fVIII immune response in hemophilic mice (Healey et al, 2007), which motivated us to analyze the frequency and epitope specificity of anti-C1 antibodies in AHA and HA inhibitor patient plasma.The frequency of domain-specific antibodies were studied by antibody binding to human A2, C1 and C2 domains presented as (i) single human domain (SHD) human/porcine hybrid fVIII and (ii) HSA-fusion proteins. While similar frequencies of A2- and C2-specific antibodies were observed for both applied mapping strategies the use of isolated C1 domain resulted in much higher detection level of anti-C1 antibodies compared to the use of the human C1 domain human/porcine hybrid fVIII protein. As homologue-scanning mutagenesis relies on differences among human and porcine sequences these results suggest the presence of a large number of cross-reactive anti-C1 antibodies binding to species-conserved epitopes. Overall, anti-C1 antibodies were detected in 90 of 115 (78%) AHA and 36 of 63 (57%) HA inhibitor patients.Two well-characterized monoclonal C1 inhibitors, human LE2E9 (Jacquemin et al, 2000) and murine MAb 2A9 (ASH 2014 poster, Batsuli et al) were used for indirect epitope mapping of anti-C1 antibodies in AHA patients by competition binding studies. Our results for AHA patients with non-crossreactive anti-C1 antibodies only (n=11) show that antibody binding to human C1 domain human/porcine hybrid fVIII (HP53) protein was completely blocked in the presence of MAb 2A9. In contrast, antibody binding to the isolated C1 domain was only partially reduced in the presence of MAb 2A9 for a selected number of (high responding) AHA patients (n=10) suggesting the presence of a second population of crossreactive anti-C1 antibodies that exclusively bind to conserved amino acid residues.Competition binding to native and denatured fVIII and HP53 proteins revealed that MAb 2A9 and LE2E9 bind mutually exclusive to a conformational C1 epitope involving amino acid residues that are not conserved between humans and pigs. Consequently, essential binding residues were identified for both C1 inhibitors via the use of HP53 variants, in which surface exposed non-conserved amino acid residues on the human C1 domain were substituted for porcine residues. The results of this mutational analysis showed that despite their competitive binding different amino acid residues are essential for binding of MAb 2A9 and LE2E9. These findings are in agreement with the different specific inhibitory activities of the two C1 inhibitors (97 BU/mg vs 10000 BU/mg).Finally, HSA-C1 point mutants were used to directly map essential epitope residues of anti-C1 antibodies in AHA and HA inhibitor patient plasma.Our study demonstrates that a large number of AHA and HA inhibitor patients (126 of 178; 71%) have anti-C1 antibodies that comprise at least two different populations, crossreactive and non-crossreactive to porcine fVIII. Therefore, in addition to the A2 and C2 domains, the C1 domain seems to significantly contribute to the immune response to fVIII in these patients. As recent data point toward a functional role of the fVIII C1 domain for membrane-, fX-, and von Willebrand factor-binding (Lü et al, 2011) the clinical relevance of anti-C1 antibodies should be analyzed in further studies. DisclosuresTiede:Leo Pharma: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria, Research Funding; Coachrom: Research Funding; SOBI: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Octapharma: Other: Investigator, Speakers Bureau; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Investigator, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Königs:Bayer: Research Funding, Speakers Bureau; Biotest: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Sobi: Consultancy; CSL Behring: Research Funding, Speakers Bureau; Intersero: Research Funding; NovoNordisk: Speakers Bureau.

Switching metal ion coordination and DNA Recognition in a Tandem CCHHC-type zinc finger peptide.

Neural Zinc Finger Factor-1 (NZF-1) and Myelin Transcription Factor 1 (MyT1) are two homologous nonclassical zinc finger (ZF) proteins that are involved in the development of the central nervous system (CNS). Both NZF-1 and MyT1 contain multiple ZF domains, each of which contains an absolutely conserved Cys2His2Cys motif. All three cysteines and the second histidine have been shown to coordinate Zn(II); however, the role of the first histidine remains unresolved. Using a functional form of NZF-1 that contains two ZF domains (NZF-1-F2F3), mutant proteins in which each histidine was sequentially mutated to a phenylalanine were prepared to determine the role(s) of the histidine residues in DNA recognition. When the first histidine is mutated, the protein binds Zn(II) in an analogous manner to the native protein. Surprisingly, this mutant does not bind to target DNA (β-RAR), suggesting that the noncoordinating histidine is critical for sequence selective DNA recognition. The first histidine will coordinate Zn(II) when the second histidine is mutated; however, the overall fold of the protein is perturbed leading to abrogation of DNA binding. NZF-1-F2F3 selectively binds to a specific DNA target sequence (from β-RAR) with high affinity (nM); while its homologue MyT1 (MyT1-F2F3), which is 92% identical to NZF-1-F2F3, binds to this same DNA sequence nonspecifically. A single, nonconserved amino acid residue in NZF-1-F2F3 is shown to be responsible for this high affinity DNA binding to β-RAR. When this residue (arginine) is engineered into the MyT1-F2F3 sequence, the affinity for β-RAR DNA increases.

Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pKa analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin–BMP interactions.

Aminoglycoside Multiacetylating Activity of the Enhanced Intracellular Survival Protein from Mycobacterium smegmatis and Its Inhibition

The enhanced intracellular survival (Eis) protein improves the survival of Mycobacterium smegmatis (Msm) in macrophages and functions as the acetyltransferase responsible for kanamycin A resistance, a hallmark of extensively drug-resistant (XDR) tuberculosis, in a large number of Mycobacterium tuberculosis (Mtb) clinical isolates. We recently demonstrated that Eis from Mtb (Eis_Mtb) efficiently multiacetylates a variety of aminoglycoside (AG) antibiotics. Here, to gain insight into the origin of substrate selectivity of AG multiacetylation by Eis, we analyzed AG acetylation by Eis_Msm, investigated its inhibition, and compared these functions to those of Eis_Mtb. Even though for several AGs the multiacetylation properties of Eis_Msm and Eis_Mtb are similar, there are three major differences. (i) Eis_Msm diacetylates apramycin, a conformationally constrained AG, which Eis_Mtb cannot modify. (ii) Eis_Msm triacetylates paromomycin, which can be only diacetylated by Eis_Mtb. (iii) Eis_Msm only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis_Mtb. Several nonconserved amino acid residues lining the AG-binding pocket of Eis are likely responsible for these differences between the two Eis homologues. Specifically, we propose that because the AG-binding pocket of Eis_Msm is more open than that of Eis_Mtb, it accommodates apramycin for acetylation in Eis_Msm, but not in Eis_Mtb. We also demonstrate that inhibitors of Eis_Mtb that we recently discovered can inhibit Eis_Msm activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis_Mtb inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation.

N-Terminal Motifs in Some Plant Disease Resistance Proteins Function in Membrane Attachment and Contribute to Disease Resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

Rational Engineering of Mannosyl Binding in the Distal Glycone Subsites of Cellulomonas fimi Endo-β-1,4-mannanase: Mannosyl Binding Promoted at Subsite −2 and Demoted at Subsite −3,

To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosaccharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.

Critical Factors Determining Dimerization of Human Antizyme Inhibitor

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K(d), suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K(d) value = 1.3 microm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K(d) value of approximately 0.1 microm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.

Novel Regulation of Smad3 Oligomerization and DNA Binding by Its Linker Domain

Smad proteins are key effectors of the transforming growth factor beta (TGFbeta) signaling pathway in mammalian cells. Smads are composed of two highly structured and conserved domains called Mad homology 1 (MH1) and 2 (MH2), which are linked together by a nonconserved linker region. The recent identification of phosphorylation sites and binding sites for ubiquitin ligases in the linker regions of TGFbeta and bone morphogenetic protein (BMP) receptor-regulated Smads suggested that the linker may contribute to the regulation of Smad function by facilitating cross-talks with other signaling pathways. In the present study, we have generated and characterized novel Smad3 mutants bearing individual substitutions of conserved and nonconserved amino acid residues within a previously described transcriptionally active linker fragment. Our analysis showed that the conserved linker amino acids glutamine 222 and proline 229 play important roles in Smad functions such as homo- and hetero-oligomerization, nuclear accumulation in response to TGFbeta stimulation, and DNA binding. Furthermore, a Smad3 mutant bearing a substitution of the nonconserved amino acid asparagine 218 to alanine displayed enhanced transactivation potential relative to wild type Smad3. Finally, Smad3 P229A inhibited TGFbeta signaling when overexpressed in mammalian cells. In conclusion, our data are in line with previous studies supporting an important regulatory role of the linker region of Smads in their function as key transducers of TGFbeta signaling.