Catalytic properties of Phe41→His mutant of horseradish peroxidase expressed inE. coli

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated fromE. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for H₂O₂ cleavage and ammonium 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, H₂O₂, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2034–2038, November, 1994.     

Unravelling Hot Spots: a comprehensive computational mutagenesis study

As protein–protein interactions are critical for all biological functions, representing a large and important class of targets for human therapeutics, identification of protein–protein interaction sites and detection of specific amino acid residues that contribute to the specificity and strength of protein interactions is very important in the biochemistry field. Alanine scanning mutagenesis has allowed the discovery of energetically crucial determinants for protein association that have been defined as hot spots. Systematic experimental mutagenesis is very laborious and time-consuming to perform, and thus it is important to achieve an accurate, predictive computational methodology for alanine scanning mutagenesis, capable of reproducing the experimental mutagenesis values. Having as a basis the MM–PBSA approach first developed by Massova et al., we performed a complete study of the influence of the variation of different parameters, such as the internal dielectric constant, the solvent representation, and the number of trajectories, in the accuracy of the free energy binding differences. As a result, we present here a very simple and fast methodological approach that achieved an overall success rate of 82% in reproducing the experimental mutagenesis data.Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

NMR and Molecular Genetics as Tools for Investigating Protein Interactions in Membrane Systems

In our laboratory, we have applied the tools of nuclear magnetic resonance (NMR) spectroscopy and molecular genetics to investigate the structural and dynamic properties of membrane-associated proteins and their interactions with membrane components. There are two general classes of membrane proteins, i.e., intrinsic and peripheral ones. For the intrinsic membrane proteins, we have chosen the membranebound D-lactate dehydrogenase (D-LDH) of Escherichia coli as a model to study protein-lipid interactions in membranes. D-LDH is a respiratory enzyme of molecularweight 65, 000 containing flavin adenine dinucleotide (FAD) as a cofactor. The activity of purified D-LDH is enhanced up to 100-fold by lipids and detergents. The gene for D-LDH has been sequenced, and production of the enzyme amplified up to 300-times normal levels. We have biosynthetically incorporated 5-fluorotryptophan (5F-Trp) into D-LDH and studied the five Trp residues by ¹⁹F-NMR spectroscopy. In order to gain additional information using ¹⁹F-NMR, site-specific, oligonucleotide-directed mutagenesis has been used to insert a sixth Trp into D-LDH at various positions throughout the 571-amino acid chain. These mutant D-LDHs are being characterized biochemically and through NMR. For peripheral membrane proteins, we have chosen two periplasmic binding proteins, histidine-binding protein J (J protein) of Salmonella Typhimurium and glutamine-binding protein (GlnBP) of E. coli as models to investigate the structure-function relationship in periplasmic binding protein-mediated active transport systems. These two proteins both have molecular weights of approximately 25, 000. By using mutant J proteins and GlnBPs and site-specific, oligonucleotide-directed mutagenesis techniques, we have assigned several resonances to specific amino acid residues. We are investigating the relationship between ligand-induced conformational changes in these two proteins and their roles in the active transport of ligand across the cell membrane. We have found that a combination of isotopic labeling, biochemistry, molecular biology, and NMR is a very useful approach to investigate various interactions of membrane-associated protein systems.     

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other β-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters K_m of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the K_cat/K_m were 181.5 s⁻¹ mM⁻¹ and 197.8 s⁻¹ mM⁻¹, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C₂₀ position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.     

Exploration of cellulose surface-binding properties of Acidothermus cellulolyticus Cel5A by site-specific mutagenesis

Understanding the interactions between cellulases and cellulosic substrates is critical to the development of an efficient artificial cellulase system for conversion of biomass to sugars. We directed specific mutations to the interactive surface of the Acidothermus cellulolyticus EI endoglucanase catalytic domain. The cellulose-binding domain is not translated in these mutants. Amino acid mutations were designed either to change the surface charge of the protein or to modify the potential for hydrogen bonding with cellulose. The relationship between cellulase-to-cellulose (Avicel PH101) binding and hydrolysis activity was determined for various groupings of mutations. While a significant increase in hydrolysis activity was not observed, certain clusters of residues did significantly alter substrate binding and some interesting correlations emerged. In the future, these observations may be used to aid the design of endoglucanases with improved performance on pretreated biomass.     

In vivo labeling method of cyclosporin A with [14C-methyl]-S-adenosyl-L-methionine can evaluate a potency of cyclosporin A-producing mutant

Cyclosporin A synthetase activity ofTolypocladium inflatum can be estimated by measuring its N-methyltransferase activity. In vivo N-methyltransferase activity of cyclosporin A synthetase of cells was measured by in vivo [¹⁴C-methyl] labeling assay, which was designed for actively growing cells. After the cells were incubated with 0.025 μCi of [¹⁴C-methyl]-S-adenosyl-l-methionine, [¹⁴C-methyl] labelled cyclosporin A and its analogs inside the cells were extracted with ethylacetate and¹⁴C radioactivity of the ethylacetate extract of the cells was counted. When various mutant cells grown on agar plate medium after ultraviolet irradiation or N-methyl-N’-nitroso-guanidine treatment were applied to in vivo [¹⁴C-methyl] labeling assay, these mutants showed a broad range of in vivo N-methyltransferase activity. Poor correlation was found between in vivo N-methyltransferase activity of cyclosporin A synthetase of the mutant grown on agar plate and the actual amount of cyclosporin A production in shake-flask culture. However, when the cells grown on the shake-flask culture were applied in the in vivo [¹⁴C-methyl] labeling assay, a better correlation was resulted. In vivo N-methyltransferase activity reached the maximum value at about 150 h, and then declined quickly, but cyclosporin A was synthesized for 200 h during fermentation. Specific in vivo N-methyltransferase activity was not greatly influenced by culture age during fermentation. The major product of in vivo [¹⁴C-methyl] labeling assay was identified as cyclosporin A, and only trace amounts of other cyclosporin analogues were detected. Therefore, the results suggest that in vivo labeling method with [C¹⁴-methyl]-S-adenosyl-l-methionine can easily compare a potency of cyclosporin A-producing mutant during fermentation.     

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1–40)

A small library of rationally designed amyloid β [Aβ(1–40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1–40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid‐state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1–40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross‐β structure.     

The importance of the long type 1 copper-binding loop of nitrite reductase for structure and function

The long 15-residue type 1 copper-binding loop of nitrite reductase has been replaced with that from the cupredoxin amicyanin (7 residues). This sizable loop contraction does not have a significant effect on the spectroscopy, and therefore, the structures of both the type 1 and type 2 Cu(II) sites. The crystal structure of this variant with Zn(II) at both the type 1 and type 2 sites has been determined. The coordination geometry of the type 2 site is almost identical to that found in the wild-type protein. However, the structure of the type 1 centre changes significantly upon metal substitution, which is an unusual feature for this class of site. The positions of most of the coordinating residues are altered of which the largest difference was observed for the coordinating His residue in the centre of the mutated loop. This ligand moves away from the active site, which results in a more open metal centre with a coordinating water molecule. Flexibility has been introduced into this region of the protein. The 200 mV increase in the reduction potential of the type 1 copper site indicates that structural changes upon reduction must stabilise the cuprous form. The resulting unfavourable driving force for electron transfer between the two copper sites, and an increased reorganisation energy for the type 1 centre, contribute to the loop variant having very little nitrite reductase activity. The extended type 1 copper-binding loop of this enzyme makes a number of interactions that are important for maintaining quaternary structure.     

Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.