Crystal Structure of a Heat-resilient Phytase from Aspergillus fumigatus, Carrying a Phosphorylated Histidine

Xiang, Tao; Liu, Qun; Deacon, Ashley M.; Koshy, Matthew; Kriksunov, I.A.; Lei, Xin Gen; Hao, Quan; Thiel, Daniel

doi:10.1016/j.jmb.2004.03.057

Cited by 53 publications

(26 citation statements)

References 31 publications

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“…The fold (Figure 4) has two domains and is very similar to those of previous 3- and 6-phytases in the PDB from the bacteria E. coli [ 26 ] , Hafnia alvei [ 27 ], Klebsiella pneumoniae [ 28 ] and Yersinia kristensenii [ 27 ] and the fungi Aspergillus ficuum [ 29 ], A. fumigatus [ 30 ], A. niger [ 31 ] and Debaryomyces castellii [ 32 ] and is typical of members of the histidine acid phosphatase superfamily. The major domain on the right is composed of residues 6–28, 47–135 and 260–410 and is colored blue and grey.…”

Section: Resultsmentioning

confidence: 53%

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

et al. 2013

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The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

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Section: Resultsmentioning

confidence: 53%

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

et al. 2013

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“…E156G substitution in PP-NP ep -6A resulted in the decreased number of putative hydrogen bond between the O atom of G156 and the N atom of K153. The hydrogen bond is a major force in steadying the higher-level structure of the enzyme-protein; more hydrogen bonds may lead to greater rigidity of the enzyme-protein [53]. For the two reasons, this substitution might enhance the conformational flexibility.…”

Section: Discussionmentioning

confidence: 99%

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR

Min

et al. 2011

Appl Biochem Biotechnol

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The mutant acid phytase (phyA ( m )) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NP(ep)-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ( ep ) gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k (m) value than that of PP-NP(m)-8 (P < 0.05). In addition, the overall catalytic efficiency (k (cat)/k (m)) of the mutant one was 84% higher (P< 0.05) than that of PP-NP(m)-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

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“…Three regions, E29-S36, G157-R162, and R241-S246, were investigated as possible factors contributing to the heat resistance of the A. fumigatus phytase (Xiang et al 2004). Comparison of the amino-acid sequences of the A. fumigatus, A. niger, and A. aculeatus phytases showed that the E29-S36 and R241-S246 sequences in the A. aculeatus phytase have the greatest homology to those in the A. fumigatus phytase, which may correlate with the high thermostability of the A. aculeatus phytase.…”

Section: Discussionmentioning

confidence: 99%

A novel thermostable phytase from the fungus Aspergillus aculeatus RCEF 4894: gene cloning and expression in Pichia pastoris

Jiang

et al. 2010

World J Microbiol Biotechnol

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A novel gene of thermostable phytase, phyA, was isolated by polymerase chain reaction (PCR) techniques from Aspergillus aculeatus RCEF 4894. The fulllength phyA gene comprises 1,404 bp and encodes 467 amino-acid residues, including a 19-residue putative N-terminal signal peptide. The phytase of A. aculeatus was a novel addition to the histidine-acid phosphatase family, as evidenced by both the conserved motifs RHGXRXP and HD in the amino-acid sequence, and 3D structure models. The recombinant phytase was overexpressed in Pichia pastoris, and its specific activity reached 3,000 U mL -1 at the optimum pH of 5.5. This recombinant, thermostable phytase was able to withstand temperatures of up to 90°C for 10 min, with a loss of only 13.9% of initial enzymatic activity, and showed high activity with phytic-acid sodium salt at a pH range of 2.5-6.5. The broad pH optima and high thermostability of the phytase makes it a promising candidate for feed-pelleting applications.

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Crystal Structure of a Heat-resilient Phytase from Aspergillus fumigatus, Carrying a Phosphorylated Histidine

Cited by 53 publications

References 31 publications

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR

A novel thermostable phytase from the fungus Aspergillus aculeatus RCEF 4894: gene cloning and expression in Pichia pastoris

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