Rikkert K. Wierenga

Thioester chemistry is exploited in Nature by many CoA-dependent enzymes. However, the covalent nature of CoA attachment largely prevents the use of these enzymes in many applications. Replacing the CoA moiety with simpler, truncated fragments, such as its pantetheine (PAN) moiety, is also hampered by the lack of understanding of the function of the CoA moiety in enzymatic conversions. Herein, we describe the utilization of the enzyme (2E)-enoyl-CoA hydratase (ECH) using PAN thioesters and an activator, 3′,5′-ADP (PAP). ECH catalyzes the hydration of the carbon–carbon double bond of (2E)-enoyl-CoA substrates in the β-oxidation lipid-degrading pathway. The hydration reaction is very challenging to carry out by traditional chemical synthesis, as no selective catalysts are available. Structural enzymology of ECH and its complexes with (3S)-hydroxyacyl-CoA products show that hydrogen bonds between the adenine 6-amino group of the ADP moiety of CoA and loop-2 induce a small structural change in this active site loop, tightening the NN distance between the hydrogen bond donors of the oxyanion hole from 5.2 Å (unliganded) to 4.0 Å and forming a competent oxyanion hole at the catalytic site. A structurally similar and catalytically competent oxyanion hole is observed in the complex with (3S)-hydroxyhexanoyl PAN and the activator 3′,5′-ADP, both bound at the active site. The use of 3′,5′-ADP as the activator enables the synthetic use of ECH for the hydration of a wide range of (2E)-enoyl-PAN substrates with different steric demands and functionalities. The products, 3-hydroxyacyl-PAN thioesters, were obtained in good isolated yields and excellent stereoselectivities (typically >99:<1 3S:3R). Even for acyl chains that contain reactive groups such as bromide or methyl ester functionalities at C7, no side products resulting from potentially competing cyclization could be detected in the enzymatic hydration protocol.

Collagen prolyl 4-hydroxylase (C-P4H) is an essential enzyme in collagen synthesis and known to be a potential target for drugs that prevent excess collagen formation. Currently known C-P4H inhibitors target the catalytic site of C-P4H, being analogues of 2-oxoglutarate (2OG). However, in mammalian cells there are many other 2OG-dependent dioxygenases with a highly similar catalytic domain, which limits the selective specificity of drugs targeting C-P4H activity. The peptide-substrate-binding (PSB) domain is unique for the C-P4H family and known to be important for the catalytic efficiency of C-P4H. Therefore, interfering with peptide binding to the PSB domain might allow more specific inhibition of the hydroxylation activity of C-P4Hs. We developed a robust FRET-based high-throughput screening assay (Z’ > 0.73) based on PSB-peptide interactions. This assay was used to screen a peptidomimetic library of 15614 compounds with the PSB domains of C-P4H isoforms I and II. A hit compound (OUL-PSBi-001) was identified with IC ₅₀ s of 40 µM and 62 µM for PSB-I and PSB-II, respectively. We also showed that this compound indeed inhibits the catalytic activity of the full-length CP4H-I and -II. This FRET assay provides a new strategy for finding selective inhibitors for the treatment of fibrotic diseases and cancer.

Collagen prolyl 4‐hydroxylase (C‐P4H) catalyzes the 4‐hydroxylation of Y‐prolines of the XYG‐repeat of procollagen. C‐P4Hs are tetrameric α₂β₂ enzymes. The α‐subunit provides the N‐terminal dimerization domain, the middle peptide‐substrate–binding (PSB) domain, and the C‐terminal catalytic (CAT) domain. There are three isoforms of the α‐subunit, complexed with a β‐subunit that is protein disulfide isomerase, forming C‐P4H I‐III. The PSB domain of the α‐subunit binds proline‐rich peptides, but its function with respect to the prolyl hydroxylation mechanism is unknown. An extended mode of binding of proline‐rich peptides (PPII, polyproline type‐II, conformation) to the PSB‐I domain has previously been reported for the PPG‐PPG‐PPG and P9 peptides. Crystal structures now show that peptides with the motif PxGP (PPG‐PRG‐PPG, PPG‐PAG‐PPG) (where x, at Y‐position 5, is not a proline) bind to the PSB‐I domain differently, more deeply, in the peptide‐binding groove. The latter mode of binding has previously been reported for structures of the PSB‐II domain complexed with these PxGP‐peptides. In addition, it is shown here by crystallographic binding studies that the POG‐PAG‐POG peptide (with 4‐hydroxyprolines at Y‐positions 2 and 8) also adopts the PxGP mode of binding to PSB‐I as well as to PSB‐II. Calorimetric binding studies show that the affinities of these peptides are lower for PSB‐I than for PSB‐II, with, respectively, K_D values of about 70 μM for PSB‐I and 20 μM for PSB‐II. The importance of these results for understanding the reaction mechanism of C‐P4H, in particular concerning the function of the PSB domain, is discussed.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α₂β₂ tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE–fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.

Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example.

Multifunctional enzyme, type‐1 (MFE1) catalyzes the second and third step of the β‐oxidation cycle, being, respectively, the 2E‐enoyl‐CoA hydratase (ECH) reaction (N‐terminal part, crotonase fold) and the NAD⁺‐dependent, 3S‐hydroxyacyl‐CoA dehydrogenase (HAD) reaction (C‐terminal part, HAD fold). Structural enzymological properties of rat MFE1 (RnMFE1) as well as of two of its variants, namely the E123A variant (a glutamate of the ECH active site is mutated into alanine) and the BCDE variant (without domain A of the ECH part), were studied, using as substrate 3S‐hydroxybutanoyl‐CoA. Protein crystallographic binding studies show the hydrogen bond interactions of 3S‐hydroxybutanoyl‐CoA as well as of its 3‐keto, oxidized form, acetoacetyl‐CoA, with the catalytic glutamates in the ECH active site. Pre‐steady state binding experiments with NAD⁺ and NADH show that the k_on and k_off rate constants of the HAD active site of monomeric RnMFE1 and the homologous human, dimeric 3S‐hydroxyacyl‐CoA dehydrogenase (HsHAD) for NAD⁺ and NADH are very similar, being the same as those observed for the E123A and BCDE variants. However, steady state and pre‐steady state kinetic data concerning the HAD‐catalyzed dehydrogenation reaction of the substrate 3S‐hydroxybutanoyl‐CoA show that, respectively, the k_cat and k_chem rate constants for conversion into acetoacetyl‐CoA by RnMFE1 (and its two variants) are about 10 fold lower as when catalyzed by HsHAD. The dynamical properties of dehydrogenases are known to be important for their catalytic efficiency, and it is discussed that the greater complexity of the RnMFE1 fold correlates with the observation that RnMFE1 is a slower dehydrogenase than HsHAD.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α ₂ β ₂ tetrameric enzyme in which the α-chain harbors the 2 E -enoyl-CoA hydratase (ECH) and 3 S -hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium, and long chain 2 E -enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modelling studies have identified binding sites for the acyl-CoA substrates at the three active sites as well as the NAD ⁺ binding pocket at the HAD active site. These studies have also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, in a crystallographic fragment binding study with MtTFE crystals 226 fragments were screened, resulting in the structures of 17 MtTFE-fragment complexes. Analysis of the 143 fragment binding events shows that the ECH active site is the ‘binding hotspot’ for the tested fragments, with 50 binding events. The mode of binding of the fragments bound at the active sites provides additional insight on how the long chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 24 fragment binding events between the active sites identify potential transient binding sites of reaction intermediates relevant for possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of these binding sites and to identify substrates for which channeling is crucial. Synopsis: Crystallographic fragment binding studies of the Mycobacterium tuberculosis trifunctional enzyme (MtTFE) have resulted in 143 binding events of 17 fragments out of 226 investigated fragments, suggesting functional sites with respect to substrate binding and substrate channeling.

Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: ( a) a nucleophilic cysteine, which forms a covalent intermediate, and ( b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.