Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.
Title: Surface-entropy reduction used in the crystallization of human choline acetyltransferase Kim AR, Dobransky T, Rylett RJ, Shilton BH Ref: Acta Crystallographica D Biol Crystallogr, 61:1306, 2005 : PubMed
Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.
Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.
Title: Crystal structure of Kex1deltap, a prohormone-processing carboxypeptidase from Saccharomyces cerevisiae Shilton BH, Thomas DY, Cygler M Ref: Biochemistry, 36:9002, 1997 : PubMed
Kex1p is a prohormone-processing serine carboxypeptidase found in Saccharomyces cerevisiae. In contrast to yeast serine carboxypeptidase (CPD-Y) and wheat serine carboxypeptidase II (CPDW-II), Kex1p displays a very narrow specificity for lysyl or arginyl residues at the C-terminus of the substrate. The structure of Kex1Deltap, an enzyme that lacks the acidic domain and membrane-spanning portion of Kex1p, has been solved by a combination of molecular replacement and multiple isomorphous replacement and refined to a resolution of 2.4 A. The S1' site of Kex1Deltap is sterically restricted compared to those from CPD-Y or CPDW-II; it also contains two acidic groups that are well positioned to interact with the basic group of a lysine or arginine side chain. The high specificity of Kex1p can therefore be explained by a combination of steric and electronic factors. The structure of the S1 site of Kex1Deltap is also well suited for binding of a lysine or arginine side chain, and the enzyme may therefore exhibit a preference for these residues at P1.
Title: Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae Shilton BH, Li Y, Tessier D, Thomas DY, Cygler M Ref: Protein Science, 5:395, 1996 : PubMed
A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.
