Enzymatic Mechanism of Acyl Transfer

Cellular proteins are often modified with chemical moieties such as phosphate or methyl groups in order to alter the function of these proteins in response to changes in the environment or during the cell cycle. Two of these post-translational modifications, acetylation and ubiquitination, involve a reaction of a thioester with an amine. While the reaction chemistry is straightforward, the enzymatic mechanism of transfer is not clear and there are a variety of protein structures that can do this chemistry. We aim to determine the catalytic mechanism of these enzymes via biochemical and structural methods to understand:

• how substrates bind

• the enzyme increases the rate of the reaction

• the role of changes in the enzyme structure in the reaction chemistry

• how substrate specificity is encoded by the enzyme

Current projects are focused on ubiquitin/ubiquitin-like activating enzymes, cleavage of ubiquitin precursors, and enzymatic modification of protein lysines.

Previous work by the lab on this project:

(undergraduate authors are indicated in bold)

Padala P, Oweis W, Mashahreh B, Soudah N, Cohen-Kfir E, Todd EA, Berndsen CE, Wiener R. Novel insights into the interaction of UBA5 with UFM1 via a UFM1-interacting sequence. Sci Rep. 2017 Mar 30;7(1):508. doi: 10.1038/s41598-017-00610-0. PMID: 28360427

Young BH, Caldwell TA, McKenzie AM, Kokhan O, Berndsen CE. (2016) Characterization of the structure and catalytic activity of Legionella pneumophila VipF. Proteins. Oct;84(10):1422-30. doi: 10.1002/prot.25087. PMID: 27315603

Oweis W, Padala P, Hassouna F, Cohen-Kfir E, Gibbs DR, Todd EA, Berndsen CE, Wiener R. (2016) Trans-Binding Mechanism of Ubiquitin-like Protein Activation Revealed by a UBA5-UFM1 Complex. Cell Rep. Sep 20;16(12):3113-20. doi: 10.1016/j.celrep.2016.08.067. PMID: 27653677

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Model of \(\beta\) amylase 2 bound to substrates

Structure and function of \(\beta\) amylase enzymes

In plants, \(\beta\) amylases cleave \(\alpha\) 1-4 linkages between dextrose residues in starch. This enzymatic activity produces maltose which can then fuel other processes in the plant. In Arabidopsis thaliana there are 9 \(\beta\) amylases however little is known about the structure and biochemistry of these enzyme. In collaboration with Dr. Jon Monroe in the JMU Biology Department we are working to characterize the structure and function of these crucial enzymes for plant metabolism

Previous work by the lab on this project:

(undergraduate authors are indicated in bold)

Monroe JD, Pope LE, Breault JS, Berndsen CE, Storm AR. Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of \(\beta\)-AMYLASE2 From Arabidopsis thaliana

Storm AR, Kohler MR, Berndsen CE, Monroe JD. Glutathionylation Inhibits the Catalytic Activity of Arabidopsis \(\beta\)-Amylase3 but Not That of Paralog \(\beta\)-Amylase1. Biochemistry. 2018 Feb 6;57(5):711-721. doi: 10.1021/acs.biochem.7b01274.

Monroe JD, Breault JS, Pope LE, Torres CE, Gebrejesus TB, Berndsen CE, Storm AR. Arabidopsis \(\beta\)-amylase2 is a K+-requiring, catalytic tetramer with sigmoidal kinetics. Plant Physiol. 2017 Oct 24. pii: pp.01506.2017. doi: 10.1104/pp.17.01506.

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Simulated pulling of human Tetherin from Ozcan and Berndsen, 2017

Viral Tethering by human Tetherin

All humans contain a simple protein known as BST-2 or Tetherin which has the innate ability to latch onto budding viruses and tether the virus to the host cell membrane. Other than a mechanical tether, there are no known functions for Tetherin and it is not exactly clear how this protein functions. We combine biophysical and computational approaches to determine the structural mechanism of viral tethering and how viral antagonists block the function of Tetherin

Support and Funding

The work of the Berndsen lab is graciously supported by:

• National Science Foundation

• Jeffress Memorial Trust for Interdisciplinary Research

• 4-VA organization

• Hamilton Syringe Company

• U.S.-Israel Binational Science Foundation