“Itching” for new strategies in protein engineering

Stephen W. Michnick; Frances H. Arnold

doi:10.1038/70699

Simulated Protein Thermal Detection (SPTD) for Enzyme Thermostability Study and an Application Example for Pullulanase from Bacillus deramificans

Current Pharmaceutical Design ◽

10.2174/1381612824666181113120948 ◽

2019 ◽

Vol 24 (34) ◽

pp. 4023-4033 ◽

Cited By ~ 7

Author(s):

Jian-Xiu Li ◽

Shu-Qing Wang ◽

Qi-Shi Du ◽

Hang Wei ◽

Xiao-Ming Li ◽

...

Keyword(s):

Protein Engineering ◽

Room Temperature ◽

Dynamics Simulation ◽

Enzyme Thermostability ◽

Different Temperatures ◽

Thermal Detection ◽

The Relationship ◽

Starch Industry ◽

Thermal Stability Of ◽

New Strategies

Background: The relationship between protein structure and its bioactivity is one of the fundamental problems for protein engineering and pharmaceutical design. Method: A new method, called SPTD (Simulated Protein Thermal Detection), was proposed for studying and improving the thermal stability of enzymes. The method was based on the evidence observed by conducting the MD (Molecular Dynamics) simulation for all the atoms of an enzyme vibrating from the velocity at a room temperature (e.g., 25°C) to the desired working temperature (e.g., 65°C). According to the recorded MD trajectories and the coordinate deviations of the constituent residues under the two different temperatures, some new strategies have been found that are useful for both drug delivery and starch industry. Conclusion: The SPTD technique presented in this paper may become a very useful tool for pharmaceutical design and protein engineering.

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New strategies of protein engineering—directed evolution of enzymein vitro

Chinese Science Bulletin ◽

10.1007/bf03183480 ◽

1999 ◽

Vol 44 (18) ◽

pp. 1641-1648 ◽

Cited By ~ 3

Author(s):

Hongying Zhang ◽

Xiangduo Kong ◽

Jin Zhang

Keyword(s):

Protein Engineering ◽

Directed Evolution ◽

New Strategies

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Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

Biochemical Journal ◽

10.1042/bcj20190812 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3835-3847 ◽

Cited By ~ 1

Author(s):

Aliyath Susmitha ◽

Kesavan Madhavan Nampoothiri ◽

Harsha Bajaj

Keyword(s):

Protein Engineering ◽

Cell Attachment ◽

Functional Characterization ◽

Protein Structures ◽

Structural Similarity ◽

Surface Proteins ◽

Sortase A ◽

Peptide Cleavage ◽

New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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