Structural enzymology studies with the substrate 3S-hydroxybutanoyl-CoA: bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD.

Multifunctional enzyme, type-1 (MFE1) catalyzes the second and third step of the ß-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 kon and koff 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 kcat and kchem 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.

Enzymes of the crotonase superfamily: Diverse assembly and diverse function.

The crotonase fold is generated by a framework of four repeats of a ßßα-unit, extended by two helical regions. The active site of crotonase superfamily (CS) enzymes is located at the N-terminal end of the helix of the third repeat, typically being covered by a C-terminal helix. A major subset of CS-enzymes catalyzes acyl-CoA-dependent reactions, allowing for a diverse range of acyl-tail modifications. Most of these enzymes occur as trimers or hexamers (dimers of trimers), but monomeric forms are also observed. A common feature of the active sites of CS-enzymes is an oxyanion hole, formed by two peptide-NH hydrogen bond donors, which stabilises the negatively charged thioester oxygen atom of the reaction intermediate. Structural properties and possible use of these enzymes for biotechnological applications are discussed.

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity.

The Fe2+-dependent E. coli enzyme FucO catalyzes the reversible interconversion of short-chain (S)-lactaldehyde and (S)-1,2-propanediol, using NADH and NAD+ as cofactors, respectively. Laboratory-directed evolution experiments have been carried out previously using phenylacetaldehyde as the substrate for screening catalytic activity with bulky substrates, which are very poorly reduced by wild-type FucO. These experiments identified the N151G/L259V double mutant (dubbed DA1472) as the most active variant with this substrate via a two-step evolutionary pathway, in which each step consisted of one point mutation. Here the crystal structures of DA1472 and its parent D93 (L259V) are reported, showing that these amino acid substitutions provide more space in the active site, though they do not cause changes in the main-chain conformation. The catalytic activity of DA1472 with the physiological substrate (S)-lactaldehyde and a series of substituted phenylacetaldehyde derivatives were systematically quantified and compared with that of wild-type as well as with the corresponding point-mutation variants (N151G and L259V). There is a 9000-fold increase in activity, when expressed as kcat/KM values, for DA1472 compared with wild-type FucO for the phenylacetaldehyde substrate. The crystal structure of DA1472 complexed with a non-reactive analog of this substrate (3,4-dimethoxyphenylacetamide) suggests the mode of binding of the bulky group of the new substrate. These combined structure-function studies therefore explain the dramatic increase in catalytic activity of the DA1472 variant for bulky aldehyde substrates. The structure comparisons also suggest why the active site in which Fe2+ is replaced by Zn2+ is not able to support catalysis.

The Role of Asn11 in Catalysis by Triosephosphate Isomerase.

Four catalytic amino acids at triosephosphate isomerase (TIM) are highly conserved: N11, K13, H95, and E167. Asparagine 11 is the last of these to be characterized in mutagenesis studies. The ND2 side chain atom of N11 is hydrogen bonded to the O-1 hydroxyl of enzyme-bound dihydroxyacetone phosphate (DHAP), and it sits in an extended chain of hydrogen-bonded side chains that includes T75' from the second subunit. The N11A variants of wild-type TIM from Trypanosoma brucei brucei (TbbTIM) and Leishmania mexicana (LmTIM) undergo dissociation from the dimer to monomer under our assay conditions. Values of Kas = 8 × 103 and 1 × 106 M-1, respectively, were determined for the conversion of monomeric N11A TbbTIM and LmTIM into their homodimers. The N11A substitution at the variant of LmTIM previously stabilized by the E65Q substitution gives the N11A/E65Q variant that is stable to dissociation under our assay conditions. The X-ray crystal structure of N11A/E65Q LmTIM shows an active site that is essentially superimposable on that for wild-type TbbTIM, which also has a glutamine at position 65. A comparison of the kinetic parameters for E65Q LmTIM and N11A/E65Q LmTIM-catalyzed reactions of (R)-glyceraldehyde 3-phosphate (GAP) and (DHAP) shows that the N11A substitution results in a (13-14)-fold decrease in kcat/Km for substrate isomerization and a similar decrease in kcat for DHAP but only a 2-fold decrease in kcat for GAP.

Structural basis for different membrane-binding properties of E. coli anaerobic and human mitochondrial ß-oxidation trifunctional enzymes.

Facultative anaerobic bacteria such as Escherichia coli have two α2ß2 heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the ß-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-ß as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-ß is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-ß dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities.

Thiolase: A Versatile Biocatalyst Employing Coenzyme A-Thioester Chemistry for Making and Breaking C-C Bonds.

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.

An engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration.

Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory function. MtFAS generates the octanoic acid precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. The structurally well-characterized component of mtFAS, human 2E-enoyl-ACP reductase (MECR) rescues respiratory growth and lipoylation defects of a Saccharomyces cerevisiae Δetr1 strain lacking native mtFAS enoyl reductase. To address the role of longer products of mtFAS, we employed in silico molecular simulations to design a MECR variant with a shortened substrate binding cavity. Our in vitro and in vivo analyses indicate that the MECR G165Q variant allows synthesis of octanoyl groups but not long chain fatty acids, confirming the validity of our computational approach to engineer substrate length specificity. Furthermore, our data imply that restoring lipoylation in mtFAS deficient yeast strains is not sufficient to support respiration and that long chain acyl-ACPs generated by mtFAS are required for mitochondrial function.

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2ß2 tetramer.

Collagen prolyl 4-hydroxylases (C-P4H) are α2ß2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the ß-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the ß/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact ß/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b', and a' domains of the ß/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a ß-strand, which is the edge strand of its major antiparallel ß-sheet. This extra region of the CAT domain interacts tightly with the ß/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a' domain and tight hydrophobic interactions with the b' domain. Using this new information, the structure of the mature C-P4H-II α2ß2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.

Substrate specificity and conformational flexibility properties of the Mycobacterium tuberculosis ß-oxidation trifunctional enzyme.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme. The α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) activity. Enzyme kinetic data reported here show that medium and long chain enoyl-CoA molecules are preferred substrates for MtTFE. Modelling studies indicate how the linear medium and long acyl chains of these substrates can bind to each of the active sites. In addition, crystallographic binding studies have identified three new CoA binding sites which are different from the previously known CoA binding sites of the three TFE active sites. Structure comparisons provide new insights into the properties of ECH, HAD and KAT active sites of MtTFE. The interactions of the adenine moiety of CoA with loop-2 of the ECH active site cause a conformational change of this loop by which a competent ECH active site is formed. The NAD+ binding domain (domain C) of the HAD part of MtTFE has only a few interactions with the rest of the complex and adopts a range of open conformations, whereas the A-domain of the ECH part is rigidly fixed with respect to the HAD part. Two loops, the CB1-CA1 region and the catalytic CB4-CB5 loop, near the thiolase active site and the thiolase dimer interface, have high B-factors. Structure comparisons suggest that a competent and stable thiolase dimer is formed only when complexed with the α-chains, highlighting the importance of the assembly for the proper functioning of the complex.

IceBear: an intuitive and versatile web application for research-data tracking from crystallization experiment to PDB deposition.

The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank.

Structure of transmembrane prolyl 4-hydroxylase reveals unique organization of EF and dioxygenase domains.

Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a third P4H-the poorly characterized endoplasmic reticulum-localized transmembrane prolyl 4-hydroxylase (P4H-TM)-is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome, but how these variants might contribute to disease is unknown. Here, we explored this question in a structural and functional analysis of soluble human P4H-TM. The crystal structure revealed an EF domain with two Ca2+-binding motifs inserted within the catalytic domain. A substrate-binding groove was formed between the EF domain and the conserved core of the catalytic domain. The proximity of the EF domain to the active site suggests that Ca2+ binding is relevant to the catalytic activity. Functional analysis demonstrated that Ca2+-binding affinity of P4H-TM is within the range of physiological Ca2+ concentration in the endoplasmic reticulum. P4H-TM was found both as a monomer and a dimer in the solution, but the monomer-dimer equilibrium was not regulated by Ca2+. The catalytic site contained bound Fe2+ and N-oxalylglycine, which is an analogue of the cosubstrate 2-oxoglutarate. Comparison with homologous P4H structures complexed with peptide substrates showed that the substrate-interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM, whereas the extensive loop structures that surround the substrate-binding groove, generating a negative surface potential, are different. Analysis of the structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with other P4Hs while having a unique EF domain.

Insights into the stability and substrate specificity of the E. coli aerobic ß-oxidation trifunctional enzyme complex.

Degradation of fatty acids by the ß-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli, the last three steps of the ß-oxidation are catalyzed by two heterotetrameric α2ß2 enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the ß-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-ß is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of ß-oxidation in E. coli.

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic ß-oxidation trifunctional enzyme complexes.

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid ß-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

The peroxisomal zebrafish SCP2-thiolase (type-1) is a weak transient dimer as revealed by crystal structures and native mass spectrometry.

The SCP2 (sterol carrier protein 2)-thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the ß-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric thiolases has previously been poorly characterized. High-resolution crystal structures of the zebrafish SCP2-thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerized monomeric form has also been captured in the obtained crystals. Four loops at the dimer interface adopt very different conformations in the monomeric form. These loops also shape the active site and their structural changes explain why a competent active site is not present in the monomeric form. Native mass spectrometry studies confirm that the zebrafish SCP2-thiolase (type-1) as well as its human homolog are weak transient dimers in solution. The crystallographic binding studies reveal the mode of binding of CoA and octanoyl-CoA in the active site, highlighting the conserved geometry of the nucleophilic cysteine, the catalytic acid/base cysteine and the two oxyanion holes. The dimer interface of SCP2-thiolase (type-1) is equally extensive as in other thiolase dimers; however, it is more polar than any of the corresponding interfaces, which correlates with the notion that the enzyme forms a weak transient dimer. The structure comparison of the monomeric and dimeric forms suggests functional relevance of this property. These comparisons provide also insights into the structural rearrangements that occur when the folded inactive monomers assemble into the mature dimer.

Structural enzymology binding studies of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase (type-II): High affinity peptides have a PxGP sequence motif.

The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α2 ß2 tetramer) binds proline-rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C-P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C-terminal domain of the α subunit). The crystal structures of the PSB domain of the human C-P4H isoform II (PSB-II) complexed with and without various short proline-rich peptides are described. The comparison with the previously determined PSB-I peptide complex structures shows that the C-P4H-I substrate peptide (PPG)3 , has at most very weak affinity for PSB-II, although it binds with high affinity to PSB-I. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB-II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB-I peptide complexes. In these PSB-II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide-binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB-I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline-rich peptide sequences that bind with high affinity to PSB-II.

Crystal structure of human anterior gradient protein 3.

Oxidative protein folding in the endoplasmic reticulum is catalyzed by the protein disulfide isomerase family of proteins. Of the 20 recognized human family members, the structures of eight have been deposited in the PDB along with domains from six more. Three members of this family, ERp18, anterior gradient protein 2 (AGR2) and anterior gradient protein 3 (AGR3), are single-domain proteins which share sequence similarity. While ERp18 has a canonical active-site motif and is involved in native disulfide-bond formation, AGR2 and AGR3 lack elements of the active-site motif found in other family members and may both interact with mucins. In order to better define its function, the structure of AGR3 is required. Here, the recombinant expression, purification, crystallization and crystal structure of human AGR3 are described.

De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form: Carbon source preferences and metabolic flux redistributions.

De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway.

Structural enzymology comparisons of multifunctional enzyme, type-1 (MFE1): the flexibility of its dehydrogenase part.

Multifunctional enzyme, type-1 (MFE1) is a monomeric enzyme with a 2E-enoyl-CoA hydratase and a 3S-hydroxyacyl-CoA dehydrogenase (HAD) active site. Enzyme kinetic data of rat peroxisomal MFE1 show that the catalytic efficiencies for converting the short-chain substrate 2E-butenoyl-CoA into acetoacetyl-CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl-CoA (to the hydratase active site) and the very similar mode of binding of NAD + and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE1 are correlated. The possible importance of the conformational flexibility of MFE1 for its biocatalytic properties is discussed. Database: Structural data are available in PDB database under the accession number 5MGB.

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1.

C: Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.

Assembly of the elongated collagen prolyl 4-hydroxylase α2ß2 heterotetramer around a central α2 dimer.

Collagen prolyl 4-hydroxylase (C-P4H), an α2ß2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The ß-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/ß-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/ß-subunits locate at the far ends of the ßααß complex. The PDI/ß-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB-CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.