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Two distinct mechanisms for TIM barrel prenyltransferases in bacteria.

ABSTRACT: The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltransferase that catalyzes an intermolecular coupling. We also show that PcrB transfers geranyl and geranylgeranyl groups to glycerol-1-phosphate (G1P), making it the first known bacterial enzyme to use G1P as a substrate. Unlike MoeO5, PcrB catalyzes prenyl transfer without isomerization to give products that retain the trans-allylic bond of the prenyl donors. The TIM barrel family of PTs is unique in including enzymes that catalyze prenyl transfer by distinctly different reaction mechanisms.

SUBMITTER: Doud EH

PROVIDER: S-EPMC3033458 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Two distinct mechanisms for TIM barrel prenyltransferases in bacteria.

Doud Emma H EH Perlstein Deborah L DL Wolpert Manuel M Cane David E DE Walker Suzanne S

Journal of the American Chemical Society 20110107 5

The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltra ...[more]

PMID: 21214173

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Project description:Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (?/?)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts.Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics.Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (?G°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ?G° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics.Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

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Two distinct mechanisms for TIM barrel prenyltransferases in bacteria.

Publications

Two distinct mechanisms for TIM barrel prenyltransferases in bacteria.

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