Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

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Identification of Amino Acid Residues of the Retroviral Aspartic Proteinases Important for Substrate Specificity and Catalytic Efficiency

Aspartic Proteinases - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4615-1871-6_52 ◽

1995 ◽

pp. 399-406 ◽

Cited By ~ 1

Author(s):

C. E. Cameron ◽

H. Burstein ◽

D. Bizub-Bender ◽

T. Ridky ◽

I. T. Weber ◽

...

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Catalytic Efficiency ◽

Amino Acid Residues ◽

Aspartic Proteinases

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Efficient Synthesis of Rare Disaccharides by Engineered β-Glucosidase Based on Hydropathy Index

10.26434/chemrxiv.11942016.v1 ◽

2020 ◽

Author(s):

Kangle Niu ◽

Zhengyao Liu ◽

Yuhui Feng ◽

Tianlong Gao ◽

Zhenzhen Wang ◽

...

Keyword(s):

Amino Acid ◽

Rational Design ◽

Large Scale ◽

Catalytic Site ◽

Scale Production ◽

Total Production ◽

Amino Acid Residues ◽

Hydropathy Index ◽

Reverse Hydrolysis ◽

Hydrolysis Activity

Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis, especially rare disaccharides, is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (<a>195.8 mg/ml/mg enzyme</a>) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, <a>TrCel1b</a>N240I lost reverse hydrolysis activity. The results demonstrate that<a> </a><a>the average hydropathy index</a> of <a>the key amino acid residues </a>in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

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