A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase

Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.     

A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family

We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5-7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.     

A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme

Using site-directed mutagenesis, the NADH-linked lactate dehydrogenase from Bacillus stearothermophilus has been specifically altered at a single residue to shift the coenzyme specificity towards NADPH. The single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine. This substitution was made to reduce steric hindrance on binding of the extra phosphate group of NADPH and to remove the negative charge of the aspartate group. The resultant mutant enzyme is 20 times more catalytically efficient than the wild-type enzyme with NADPH.     

Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement

To improve the thermostability and catalytic activity of Aspergillus niger xylanase A (AnxA), its N-terminus was substituted with the corresponding region of Thermomonospora fusca xylanase A (TfxA). The constructed hybrid xylanase, named ATx, was overexpressed in Pichia pastoris and secreted into the medium. After 96-h 0.25% methanol induction, the activity of the ATx in the culture supernatant reached its peak, 633 U/mg, which was 3.6 and 5.4 times as high as those of recombinant AnxA (reAnxA) and recombinant TfxA (reTfxA), respectively. Studies on enzymatic properties showed that the temperature and pH optimum of the ATx were 60 degrees C and 5.0, respectively. The ATx was more thermostable, when it was treated at 70 degrees C, pH 5.0, for 2 min, the residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The ATx was very stable over a broader pH range (3.0-10.0) and much less affected by acid/base conditions. After incubation at pH 3.0-10.0, 25 degrees C for 1 h, all the residual activities of the ATx were over 80%. These results revealed that the thermostability and catalytic activity of the AnxA were enhanced. The N-terminus of TfxA contributed to the observed thermostability of itself and the ATx, and to the high activity of the ATx. Replacement of N-terminus between mesophilic eukaryotic and thermostable prokaryotic enzymes may be a useful method for constructing the new and improved versions of biologically active enzymes.     

Enhancing catalytic activity of a hybrid xylanase through single substitution of Leu to Pro near the active site

A modified error-prone PCR and high-throughout screening system based on 96-well plate were employed to improve catalytic activity of a hybrid xylanase (ATx). The mutant (FSI-A124) with enhanced activity was further heterologously expressed in Pichia pastoris under the control of GAP promoter. The recombinant xylanase driven by the Saccharomyces cerevisiae α-mating factor was secreted into culture medium. After growth in YPD medium for 96 h, xylanase activity in the culture supernatant reached 66.1 U ml⁻¹, which was 2.92 times as that of its parent. 6 × His-tagged purification increased the specific activity to 1557.61 U mg⁻¹. The optimum temperature and pH of recombinant xylanase were 55°C and 6.0, respectively. A single amino acid substitution (L49P) was observed within sequence of the mutant. Insight of the three dimensional structure revealed that proline possibly produced weaker hydrogen bond, van der Waals force and hydrophobic interaction with other residues nearby than leucine, especially for V174, contributing to the flexibility of catalytic residue E177. In this study, FSI-A124 exhibited higher xylanase activity but poorer thermostability than its parent, indicating that activity and stability might be negatively correlated.     

A single amino acid substitution in the beta-adrenergic receptor promotes partial agonist activity from antagonists

The family of G-protein-linked receptors includes many important pharmacological targets, of which the beta-adrenergic receptor is one of the best characterized. A better understanding of those factors that determine whether a ligand functions as an antagonist or as an agonist would facilitate the development of pharmaceutical agents that act at these receptors. Site-directed mutagenesis of the hamster beta 2-adrenergic receptor has implicated the conserved Asp113 residue in the third hydrophobic domain of the receptor in the interaction with cationic amine agonists and antagonists (Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) J. Biol. Chem, 263, 10267-10271). We now report that substitution of Asp113 with a glutamic acid residue results in a mutant beta-adrenergic receptor which recognizes several known beta-adrenergic antagonists as partial agonists. This partial agonist activity requires the presence of a carboxylate side chain on the amino acid residue at position 113 and is not observed when an asparagine residue is substituted at this position. These observations support the existence of overlapping binding sites for agonists and antagonists on the beta-adrenergic receptor and demonstrate that genetic engineering of receptors can complement structure-activity studies of ligands in defining the molecular interactions involved in receptor activation.     

An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation

The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2₁2₁2₁ and the structure was determined at a resolution of 2.1 Å. The enzyme has the common eight-fold TIM-barrel structure of family 10 xylanases; however, unlike non-alkaline active xylanases, it has a highly negatively charged surface and a deeper active site cleft. Mutational analysis of non-conserved amino acids which are close to the acid/base residue has shown that Val169, Ile170 and Asp171 are important to hydrolyze xylan at high pH. Unlike the wild type xylanase which has optimum pH at 9–9.5, the triple mutant xylanase (V169A, I170F and D171N), which was constructed using sequence information of alkaline sensitive xylanses was optimally active around pH 7. Compared to non-alkaline active xylanases, the alkaline active xylanases have highly acidic surfaces and fewer solvent exposed alkali labile residues. Based on these results obtained from sequence, structural and mutational analysis, the possible mechanisms of high pH stability and catalysis are discussed. This will provide useful information to understand the mechanism of high pH adaptation and engineering of enzymes that can be operationally stable at high pH.     

Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution

Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 A away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine. The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature (Tm) of 43 degrees C, whereas the wild-type AP dimer has a Tm of 97 degrees C. The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.     

A single amino acid substitution alters ClpS2 binding specificity

ClpS2 is a small protein under development as a probe for selectively recognizing N-terminal amino acids of N-degron peptide fragments. To understand the structural basis of ClpS2 specificity for an N-terminal amino acid, all atom molecular dynamics (MD) simulations were conducted using the sequence of a bench-stable mutant of ClpS2, called PROSS. We predicted that a single amino acid leucine to asparagine substitution would switch the specificity of PROSS ClpS2 to an N-terminal tyrosine over the preferred phenylalanine. Experimental validation of the mutant using a fluorescent yeast-display assay showed an increase in tyrosine binding over phenylalanine, in support of the proposed hypothesis.     

Structural destabilization of the recombinant thermophilic TthL11 ribosomal protein by a single amino acid substitution

Thermus thermophilus L11 protein has previously been reported to be resistant against tryptic and chymotryptic proteolysis under native conditions. With a single amino acid substitution, namely Trp101Arg, conformational changes were induced that resulted in the exhibition of specific amino acids that served as targets for tryptic and chymotryptic action and rendered the protein highly unstable even during purification. This unexpected process was evidenced by the isolation with size exclusion gel chromatography of the well-structured chymotryptic N-terminal domain in a high amount and its characterization both by Edman degradation and QTOF-EMS spectroscopy. On the other hand, the substitution of Val38Cys, which did not contribute to structural changes, indicates a very possible implication of this amino acid in the protein methylation process. The data reported in this work illustrate the distinctive amino acid dynamics in a thermophilic protein, which, while serving the function common to its counterparts from mesophilic organisms, has had to adapt to the extreme environmental conditions typical of thermophilic organisms.     

A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA

Dnmt3a and Dnmt3b are paralogous enzymes responsible for de novo DNA methylation but with distinguished biological functions. In mice, disruption of Dnmt3b but not Dnmt3a causes global DNA hypomethylation, especially in repetitive sequences, which comprise the large majority of methylated DNA in the genome. By measuring DNA methylation activity of Dnmt3a and Dnmt3b homologues from five species, we found that mammalian Dnmt3b possessed significantly higher methylation activity on chromatin DNA than Dnmt3a and non-mammalian Dnmt3b. Sequence comparison and mutagenesis experiments identified a single amino acid substitution (I662N) in mammalian Dnmt3b as being crucial for its high chromatin DNA methylation activity. Further mechanistic studies demonstrated this substitution markedly enhanced the binding of Dnmt3b to nucleosomes and hence increased the chromatin DNA methylation activity. Moreover, this substitution was crucial for Dnmt3b to efficiently methylate repetitive sequences, which increased dramatically in mammalian genomes. Consistent with our observation that Dnmt3b evolved more rapidly than Dnmt3a during the emergence of mammals, these results demonstrated that the I662N substitution in mammalian Dnmt3b conferred enhanced chromatin DNA methylation activity and contributed to functional adaptation in the epigenetic system.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.     

Inferring natural selection operating on conservative and radical substitution at single amino acid sites

Natural selection operating on amino acid substitution at single amino acid sites can be detected by comparing the rates of synonymous (r(S)) and nonsynonymous (r(N)) nucleotide substitution at single codon sites. Amino acid substitutions can be classified as conservative or radical according to whether they retain the properties of the substituted amino acid. Here methods for comparing the rates of conservative (r(C)) and radical (r(R)) nonsynonymous substitution with r(S) at single codon sites were developed to detect natural selection operating on these substitutions at single amino acid sites. A method for comparing r(C) and r(R) at single codon sites was also developed to detect biases toward these substitutions at single amino acid sites. Charge was used as the property of the amino acids. In a computer simulation, false-positive rates of these methods were always < 5%, unless termination sites were included in the computation of the numbers of sites and estimates of transition/transversion rate ratio were highly biased. The frequency of detection of natural selection operating on conservative substitution was almost independent of the presence of natural selection operating on radical substitution, and vice versa. Natural selection operating specifically on conservative and radical substitution was detected more efficiently by comparing r(S) with r(C) and r(S) with r(R) than by comparing r(S) with r(N). These methods also appeared to be robust against the occurrence of recombination during evolution. In an analysis of class I human leukocyte antigen, negative selection operating on conservative substitution, but not positive selection operating on radical substitution, was observed at some of the codon sites with r(R) > r(C), suggesting that r(R) > r(C) may not necessarily be an indicator of positive selection operating on radical substitution.     

A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.