Dataset Information

Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery.

ABSTRACT: FAT10 conjugation, a post-translational modification analogous to ubiquitination, specifically requires UBA6 and UBE2Z as its activating (E1) and conjugating (E2) enzymes. Interestingly, these enzymes can also function in ubiquitination. We have determined the crystal structure of UBE2Z and report how the different domains of this E2 enzyme are organized. We further combine our structural data with mutational analyses to understand how specificity is achieved in the FAT10 conjugation pathway. We show that specificity toward UBA6 and UBE2Z lies within the C-terminal CYCI tetrapeptide in FAT10. We also demonstrate that this motif slows down transfer rates for FAT10 from UBA6 onto UBE2Z.

SUBMITTER: Schelpe J

PROVIDER: S-EPMC4705383 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery.

Schelpe Julien J Monté Didier D Dewitte Frédérique F Sixma Titia K TK Rucktooa Prakash P

The Journal of biological chemistry 20151110 2

FAT10 conjugation, a post-translational modification analogous to ubiquitination, specifically requires UBA6 and UBE2Z as its activating (E1) and conjugating (E2) enzymes. Interestingly, these enzymes can also function in ubiquitination. We have determined the crystal structure of UBE2Z and report how the different domains of this E2 enzyme are organized. We further combine our structural data with mutational analyses to understand how specificity is achieved in the FAT10 conjugation pathway. We ...[more]

PMID: 26555268

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Project description:Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 (CpFatB1) (C8 specificity, lower activity) and CpFatB2 (C14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify CpFatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from CpFatB1 into CpFatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control.IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Similarly, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.

Dataset Information

Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery.

Publications

Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery.

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