Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

Abstract Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3–2.4-fold, improved their stability at 60 °C and pH 1.0–2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content.

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Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin

Biochemical Journal ◽

10.1042/bj2980507 ◽

1994 ◽

Vol 298 (2) ◽

pp. 507-510 ◽

Cited By ~ 4

Author(s):

A Betz ◽

P C R Hopkins ◽

B F Le Bonniec ◽

S R Stone

Keyword(s):

Crystal Structure ◽

Binding Energy ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Ionic Interactions ◽

Directed Mutagenesis ◽

Side Chains ◽

Core Domain ◽

The Core ◽

The Stability

The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.

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N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Applied and Environmental Microbiology ◽

10.1128/aem.02881-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1004-1014 ◽

Cited By ~ 23

Author(s):

Canfang Niu ◽

Huiying Luo ◽

Pengjun Shi ◽

Huoqing Huang ◽

Yaru Wang ◽

...

Keyword(s):

Acid Phosphatase ◽

Biochemical Characterization ◽

Animal Feed ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Acidic Ph ◽

Cleavage Sites ◽

Ph Optimum ◽

Content Type ◽

Histidine Acid Phosphatase

ABSTRACTN-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases fromYersinia kristensenii(YkAPPA) andYersinia rohdei(YrAPPA), each having anN-glycosylation motif, and one pepsin-sensitive HAP phytase fromYersinia enterocolitica(YeAPPA) that lacked anN-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering theN-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed inPichia pastorisfor biochemical characterization. Compared with those of theN-glycosylation site deletion mutants andN-deglycosylated enzymes, allN-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of theN-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of theN-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced inEscherichia colibut had no effect on the pepsin resistance ofN-glycosylated enzymes produced inP. pastoris. Thus,N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation ofN-glycosylation, for improvement of phytase properties for use in animal feed.

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Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.3.543 ◽

1999 ◽

Vol 43 (3) ◽

pp. 543-548 ◽

Cited By ~ 13

Author(s):

Sonia Trépanier ◽

James R. Knox ◽

Natalie Clairoux ◽

François Sanschagrin ◽

Roger C. Levesque ◽

...

Keyword(s):

Three Dimensional ◽

Catalytic Efficiency ◽

Enterobacter Cloacae ◽

Acid Group ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Class C ◽

Three Dimensional Models ◽

Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

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Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01703-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3123-3126 ◽

Cited By ~ 5

Author(s):

Carlo Bottoni ◽

Mariagrazia Perilli ◽

Francesca Marcoccia ◽

Alessandra Piccirilli ◽

Cristina Pellegrini ◽

...

Keyword(s):

Catalytic Activity ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Zinc Ion ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

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Enhancement of enzymatic activity for myoglobins by modification of heme-propionate side chains

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424604000246 ◽

2004 ◽

Vol 08 (03) ◽

pp. 255-264 ◽

Cited By ~ 5

Author(s):

Takashi Hayashi ◽

Hideaki Sato ◽

Takashi Matsuo ◽

Takaaki Matsuda ◽

Yutaka Hitomi ◽

...

Keyword(s):

Binding Site ◽

Metal Binding ◽

Protein Function ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Side Chains ◽

Cytochrome P450 Enzyme ◽

Prosthetic Group ◽

Heme Pocket ◽

P450 Enzyme

The modification of myoglobin is an attractive process not only for understanding its molecular mechanism but also for engineering the protein function. The strategy of myoglobin functionalization can be divided into at least two approaches: site-directed mutagenesis and reconstitution with a non-natural prosthetic group. The former method enables us to mainly modulate the physiological function, while the latter has the advantage of introducing a new function on the protein. Particularly, replacement of the native hemin with an artificially created hemin having hydrophobic moieties at the terminal of the heme-propionate side chains serves as an appropriate substrate-binding site near the heme pocket, and consequently enhances the peroxidase and peroxygenase activities for the reconstituted myoglobin. In addition, the incorporation of the synthetic hemin bearing modified heme-propionates into an appropriate apomyoglobin mutant drastically enhances the peroxidase activity. In contrast, to convert myoglobin into a cytochrome P450 enzyme, a flavin moiety as an electron transfer mediator was introduced at the terminal of the heme-propionate side chain. The flavomyoglobin catalyzes the deformylation of 2-phenylpropanal in the presence of NADH under aerobic conditions through the peroxoanion formation from the oxygenated species. In addition, modification of the heme-propionate side chains has an significant influence on regulating the reactivity of the horseradish peroxidase. Furthermore, the heme-propionate side chain can form a metal binding site with a carboxylate residue in the heme pocket. These studies indicate that modification of the heme-propionate side chains can be a new and effective way to engineer functions for the hemoproteins.

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Cleavage At Arg1018 During Thrombin-Mediated Activation of Coagulation Factor V Is Dispensable

Blood ◽

10.1182/blood.v118.21.1186.1186 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1186-1186 ◽

Cited By ~ 1

Author(s):

Mahesheema Na ◽

Joesph R Wiencek ◽

Jamila Hirbawi ◽

Michael Kalafatis

Keyword(s):

Conflicts Of Interest ◽

Membrane Surface ◽

Factor Xa ◽

Coagulation Factor ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Factor V ◽

Cleavage Sites ◽

Two Stage ◽

Cofactor Activity

Abstract Abstract 1186 Blood clotting results in the proteolytic conversion of prothrombin (Pro) to thrombin which in turn will produce the fibrin clot. The proteolytic conversion of Pro to thrombin is catalyzed by the prothrombinase complex which is composed of the enzyme, factor Xa (FXa), the cofactor, factor Va (FVa), assembled on a membrane surface in the presence of divalent metal ions. Factor V (FV), is a multidomain protein (A1-A2-B-A3-C1-C2) with nominal procoagulant activity and is activated by thrombin to FVa through three sequential proteolytic cleavages at Arg709, Arg1018 and Arg1545. To understand the significance of each cleavage for active cofactor formation and prothrombinase function, recombinant factor V molecules were created by site-directed mutagenesis with two out of three cleavage sites mutated simultaneously (to glutamine). We have generated a FV molecule mutated at the Arg709/1018 cleavage sites (FVQQR), a FV molecule mutated at the Arg709/1545 cleavage sites (FVQRQ), a FV molecule mutated at the Arg1018/1545 cleavage sites (FVRQQ), and a FV molecule that is mutated at all three cleavage sites (FVQQQ). These recombinant FV molecules along with wild type FV (FVWT) were transiently expressed in COS7L cells, purified to homogeneity and assessed for their capability to interact with factor Xa following activation by thrombin, and participate in prothrombinase. Pro activation by prothrombinase assembled with the mutant molecules was evaluated by SDS-PAGE and the kinetic parameters of the reactions in the presence of saturating concentrations of FXa were determined. Two-stage clotting assays revealed that while FVQQQ was devoid of clotting activity following incubation with thrombin, FVaQQR, FVaQRQ and FVaRQQ all had impaired clotting activities compared to FVaWT and plasma derived FVa (FVaPLASMA). Kinetic analyses demonstrated that FVaWT had a Kd of 0.25nM for FXa while all other mutant molecules had impaired binding capabilities for FXa. FVaQQQ was severely impaired in its ability to interact with FXa. The kcat value for prothrombinase assembled with FVaQQR was approximately 50% lower than the kcat obtained with prothrombinase assembled with FVaWT, while prothrombinase assembled with FVaQRQ and FVaRQQ had approximately 3-fold reduced catalytic efficiency when compared to the values obtained with prothrombinase assembled with FVaWT. Following incubation with thrombin prothrombinase assembled with FVaQQQ had no cofactor activity. To determine the importance of the cleavage site at Arg1018 for procofactor activation and the function of amino acid region 1000–1008 during proteolysis, several other recombinant molecules were generated. FVRQR is a FV molecule with the mutation Arg1018→Gln, and FVΔ1000-1008 is a mutant FV molecule with region 1000–1008 deleted. We have also generated FVΔ1000-1008/RQR and FVΔ1000-1008/QRQ. Two-stage clotting assays revealed that FVaRQR and FVaΔ1000-1008/RQR have similar clotting activities as FVaWT, whereas FVaQRQ, FVaΔ1000-1008/QRQ are impaired in their clotting activities. Kinetic analyses demonstrated that FVaRQR and FVaΔ1000-1008/RQR have similar affinity for FXa as FVa WT while FVaQRQ and FVaΔ1000-1008/QRQ were impaired in their interaction with factor Xa. The kcat values for prothrombinase assembled with FVaRQR and FVaΔ1000-1008/RQR were similar to the kcat obtained with prothrombinase assembled with FVa WT, while prothrombinase assembled with FVaQRQ and FVaΔ1000-1008/QRQ had 2-fold and 7-fold reduced catalytic efficiency respectively, when compared to the kcat values obtained with prothrombinase assembled with FVaWT. Overall, the data demonstrate that cleavage at both Arg709 and Arg1545 are a prerequisite for expression of optimum cofactor activity. Our data also suggests that cleavage at Arg1018 is redundant for cofactor activity. The role of cleavage at this site by thrombin during procofactor activation remains to be determined. Disclosures: No relevant conflicts of interest to declare.

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Evolutionarily conserved structural motifs in bacterial GST (glutathione S-transferase) are involved in protein folding and stability

Biochemical Journal ◽

10.1042/bj20051367 ◽

2006 ◽

Vol 394 (1) ◽

pp. 11-17 ◽

Cited By ~ 18

Author(s):

Nerino Allocati ◽

Michele Masulli ◽

Marilena Pietracupa ◽

Luca Federici ◽

Carmine Di Ilio

Keyword(s):

Protein Folding ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glutathione S Transferase ◽

Structural Motifs ◽

Hydrogen Bond Interactions ◽

Related Organism ◽

Bonding Interaction ◽

The Stability ◽

Capping Box

The bacterium Proteus mirabilis expresses a cytosolic class beta glutathione S-transferase (PmGST B1-1) that is part of a family of multifunctional detoxication enzymes. Like other cytosolic GSTs, PmGST B1-1 possesses two local structural motifs, an N-capping box and a hydrophobic staple motif, both of which are located between amino acids 151 and 156. The N-capping box consists of a reciprocal hydrogen bonding interaction of Thr152 with Asp155, whereas the hydrophobic staple motif consists of a hydrophobic interaction between Phe151 and Ala156. By contrast with other GSTs, PmGST B1-1 displays distinct hydrogen bond interactions in the N-capping box. In mammalian GSTs these structural elements are critical for protein folding and stability. To investigate the role played by these two motifs in a distantly related organism on the evolutionary scale, site-directed mutagenesis was used to generate several mutants of both motifs in PmGST B1-1. All mutants were efficiently overexpressed and purified, but they were quite unstable, although at different levels, indicating that protein folding was significantly destabilized. The analysis of the T152A and D155G variants indicated that the N-capping box motif plays an important role in the stability and correct folding of the enzyme. The analysis of F151A and A156G mutants revealed that the hydrophobic staple motif influences the structural maintenance of the protein and is implicated in the folding process of PmGST B1-1. Finally, the replacement of Thr152 and Asp155, as well as Phe151 and Ala156 residues influences the catalytic efficiency of the enzyme.

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The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants

Biochemical Journal ◽

10.1042/bj2990347 ◽

1994 ◽

Vol 299 (2) ◽

pp. 347-350 ◽

Cited By ~ 8

Author(s):

T I Koshy ◽

T L Luntz ◽

B Plotkin ◽

A Schejter ◽

E Margoliash

Keyword(s):

Cytochrome C ◽

Baker’S Yeast ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Baker's Yeast ◽

Guanidinium Chloride ◽

Mutant Proteins ◽

Cytochromes C ◽

Structural Differences ◽

The Stability

The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1. Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol. These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.

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Surface induced self-organization of comb-like macromolecules

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.2.61 ◽

2011 ◽

Vol 2 ◽

pp. 569-584 ◽

Cited By ~ 7

Author(s):

Konstantin I Popov ◽

Vladimir V Palyulin ◽

Martin Möller ◽

Alexei R Khokhlov ◽

Igor I Potemkin

Keyword(s):

Ultrathin Films ◽

Self Assembly ◽

Self Organization ◽

Side Chain ◽

Spontaneous Curvature ◽

Side Chains ◽

Polymer Architectures ◽

Different Types ◽

Surface Domains ◽

The Stability

We present a review of the theoretical and experimental evidence for the peculiar properties of comb copolymers, demonstrating the uniqueness of these materials among other polymer architectures. These special properties include an increase in stiffness upon increasing side-chain length, the spontaneous curvature of adsorbed combs, rod–globule transition, and specific intramolecular self-assembly. We also propose a theory of chemically heterogeneous surface nanopattern formation in ultrathin films of comblike macromolecules containing two different types (A and B) of incompatible side chains (so-called binary combs). Side chains of the binary combs are strongly adsorbed on a surface and segregated with respect to the backbone. The thickness of surface domains formed by the B side chains is controlled by the interaction with the substrate. We predict the stability of direct and inverse disc-, torus- and stripelike nanostructures. Phase diagrams of the film are constructed.

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Influence of Flexible Side-Chains on the Breathing Phase Transition of Pillared Layer MOFs: A Force Field Investigation

10.26434/chemrxiv.11720679.v1 ◽

2020 ◽

Author(s):

Julian Keupp ◽

Johannes P. Dürholt ◽

Rochus Schmid

Keyword(s):

First Principles ◽

Good Accuracy ◽

Field Investigation ◽

Square Lattice ◽

Side Chain ◽

Second Step ◽

Side Chains ◽

Dynamics Simulations ◽

Complex Phenomena ◽

Closed Pore

The prototypical pillared layer MOFs, formed by a square lattice of paddle- wheel units and connected by dinitrogen pillars, can undergo a breathing phase transition by a “wine-rack” type motion of the square lattice. We studied this not yet fully understood behavior using an accurate first principles parameterized force field (MOF-FF) for larger nanocrystallites on the example of Zn 2 (bdc) 2 (dabco) [bdc: benzenedicarboxylate, dabco: (1,4-diazabicyclo[2.2.2]octane)] and found clear indi- cations for an interface between a closed and an open pore phase traveling through the system during the phase transformation [Adv. Theory Simul. 2019, 2, 11]. In conventional simulations in small supercells this mechanism is prevented by periodic boundary conditions (PBC), enforcing a synchronous transformation of the entire crystal. Here, we extend this investigation to pillared layer MOFs with flexible side-chains, attached to the linker. Such functionalized (fu-)MOFs are experimen- tally known to have different properties with the side-chains acting as fixed guest molecules. First, in order to extend the parameterization for such flexible groups, 1a new parametrization strategy for MOF-FF had to be developed, using a multi- structure force based fit method. The resulting parametrization for a library of fu-MOFs is then validated with respect to a set of reference systems and shows very good accuracy. In the second step, a series of fu-MOFs with increasing side-chain length is studied with respect to the influence of the side-chains on the breathing behavior. For small supercells in PBC a systematic trend of the closed pore volume with the chain length is observed. However, for a nanocrystallite model a distinct interface between a closed and an open pore phase is visible only for the short chain length, whereas for longer chains the interface broadens and a nearly concerted trans- formation is observed. Only by molecular dynamics simulations using accurate force fields such complex phenomena can be studied on a molecular level.

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