Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

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.

Expression and analysis of the SAM-dependent RNA methyltransferase Rsm22 from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2 kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117-Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.

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.

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment.

The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal-epidermal junction and connects the cytoplasmic keratin network to the extracellular basement membrane. Dysfunction of BP180 caused by mutations in patients with junctional epidermolysis bullosa or autoantibodies in those with bullous pemphigoid leads to severe skin blistering. The extracellular collagenous domain of BP180 participates in the protein's triple-helical folding, but the structure and functional importance of the intracellular domain (ICD) of BP180 are largely unknown. In the present study, we purified and characterized human BP180 ICD. When expressed in Escherichia coli as glutathione-S-transferase or 6 × histidine tagged fusion protein, the BP180 ICD was found to exist as a monomer. Analysis of the secondary structure content by circular dichroism spectroscopy revealed that the domain is intrinsically disordered. This finding aligned with that of a bioinformatic analysis, which predicted a disordered structure. Interestingly, both anionic detergent micelles and lipid vesicles induced partial folding of the BP180 ICD, suggesting that in its natural environment, the domain's folding and unfolding may be regulated by interaction with the cell membrane or accompanying proteins. We hypothesize that the intrinsically disordered structure of the ICD of BP180 contributes to the mechanism that allows the remodeling of hemidesmosome assembly.

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.

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.

The extended structure of the periplasmic region of CdsD, a structural protein of the type III secretion system of Chlamydia trachomatis.

The type III secretion system (T3SS) is required for the virulence of many gram-negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM-ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C-terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αßßαß topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C-terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small-angle X-ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24-mer complex forming the outer ring of the IM-ring of the T3SS.

Structures of yeast peroxisomal Δ(3),Δ(2)-enoyl-CoA isomerase complexed with acyl-CoA substrate analogues: the importance of hydrogen-bond networks for the reactivity of the catalytic base and the oxyanion hole.

Δ(3),Δ(2)-Enoyl-CoA isomerases (ECIs) catalyze the shift of a double bond from 3Z- or 3E-enoyl-CoA to 2E-enoyl-CoA. ECIs are members of the crotonase superfamily. The crotonase framework is used by many enzymes to catalyze a wide range of reactions on acyl-CoA thioesters. The thioester O atom is bound in a conserved oxyanion hole. Here, the mode of binding of acyl-CoA substrate analogues to peroxisomal Saccharomyces cerevisiae ECI (ScECI2) is described. The best defined part of the bound acyl-CoA molecules is the 3',5'-diphosphate-adenosine moiety, which interacts with residues of loop 1 and loop 2, whereas the pantetheine part is the least well defined. The catalytic base, Glu158, is hydrogen-bonded to the Asn101 side chain and is further hydrogen-bonded to the side chain of Arg100 in the apo structure. Arg100 is completely buried in the apo structure and a conformational change of the Arg100 side chain appears to be important for substrate binding and catalysis. The oxyanion hole is formed by the NH groups of Ala70 (loop 2) and Leu126 (helix 3). The O atoms of the corresponding peptide units, Gly69 O and Gly125 O, are both part of extensive hydrogen-bond networks. These hydrogen-bond networks are a conserved feature of the crotonase oxyanion hole and their importance for catalysis is discussed.

Human Δ³,Δ²-enoyl-CoA isomerase, type 2: a structural enzymology study on the catalytic role of its ACBP domain and helix-10.

The catalytic domain of the trimeric human Δ(3),Δ(2)-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the ß-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis.

The structural motifs for substrate binding and dimerization of the α subunit of collagen prolyl 4-hydroxylase.

Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the proline hydroxylation of procollagen, an essential modification in the maturation of collagens. C-P4H consists of two catalytic α subunits and two protein disulfide isomerase ß subunits. The assembly of these subunits is unknown. The α subunit contains an N domain (1-143), a peptide-substrate-binding-domain (PSB, 144-244) and a catalytic domain (245-517). Here, we report the dimeric structure of the N-terminal region (1-244) of the α subunit. It is shown that the N domain has an important role in the assembly of the C-P4H tetramer, by forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two α subunits. Complexes of this construct with a C-P4H inhibitor and substrate show the mode of peptide-binding to the PSB domain. Both peptides adopt a poly-(L)-proline-type-II helix conformation and bind in a curved, asymmetric groove lined by conserved tyrosines and an Arg-Asp salt bridge.

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics.

All of the peroxisomal ß-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type 2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.

The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif.

Plant and algal prolyl 4-hydroxylases (P4Hs) are key enzymes in the synthesis of cell wall components. These monomeric enzymes belong to the 2-oxoglutarate dependent superfamily of enzymes characterized by a conserved jelly-roll framework. This algal P4H has high sequence similarity to the catalytic domain of the vertebrate, tetrameric collagen P4Hs, whereas there are distinct sequence differences with the oxygen-sensing hypoxia-inducible factor P4H subfamily of enzymes. We present here a 1.98-A crystal structure of the algal Chlamydomonas reinhardtii P4H-1 complexed with Zn(2+) and a proline-rich (Ser-Pro)(5) substrate. This ternary complex captures the competent mode of binding of the peptide substrate, being bound in a left-handed (poly)l-proline type II conformation in a tunnel shaped by two loops. These two loops are mostly disordered in the absence of the substrate. The importance of these loops for the function is confirmed by extensive mutagenesis, followed up by enzyme kinetic characterizations. These loops cover the central Ser-Pro-Ser tripeptide of the substrate such that the hydroxylation occurs in a highly buried space. This novel mode of binding does not depend on stacking interactions of the proline side chains with aromatic residues. Major conformational changes of the two peptide binding loops are predicted to be a key feature of the catalytic cycle. These conformational changes are probably triggered by the conformational switch of Tyr(140), as induced by the hydroxylation of the proline residue. The importance of these findings for understanding the specific binding and hydroxylation of (X-Pro-Gly)(n) sequences by collagen P4Hs is also discussed.

The active site of an algal prolyl 4-hydroxylase has a large structural plasticity.

Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90 A resolution, one complexed with the competitive inhibitor Zn2+, and one with Zn2+ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85 A resolution. The structures reveal the double-stranded beta-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystallographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxia-inducible factor P4Hs use proline-rich peptides as their substrates.

Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis.

D-bifunctional protein (DBP) deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. The clinical presentation of DBP deficiency is usually very severe, but a few patients with a relatively mild presentation have been identified. In this article, we report the mutational spectrum of DBP deficiency on the basis of molecular analysis in 110 patients. We identified 61 different mutations by DBP cDNA analysis, 48 of which have not been reported previously. The predicted effects of the different disease-causing amino acid changes on protein structure were determined using the crystal structures of the (3R)-hydroxyacyl-coenzyme A (CoA) dehydrogenase unit of rat DBP and the 2-enoyl-CoA hydratase 2 unit and liganded sterol carrier protein 2-like unit of human DBP. The effects ranged from the replacement of catalytic amino acid residues or residues in direct contact with the substrate or cofactor to disturbances of protein folding or dimerization of the subunits. To study whether there is a genotype-phenotype correlation for DBP deficiency, these structure-based analyses were combined with extensive biochemical analyses of patient material (cultured skin fibroblasts and plasma) and available clinical information on the patients. We found that the effect of the mutations identified in patients with a relatively mild clinical and biochemical presentation was less detrimental to the protein structure than the effect of mutations identified in those with a very severe presentation. These results suggest that the amount of residual DBP activity correlates with the severity of the phenotype. From our data, we conclude that, on the basis of the predicted effect of the mutations on protein structure, a genotype-phenotype correlation exists for DBP deficiency.

Structural biology of the thioester-dependent degradation and synthesis of fatty acids.

The fatty acid degradation and synthesis pathways consist of the same four chemical transformations. These transformations are facilitated by conjugating the fatty acid, via a thioester bond, to coenzyme A or acyl carrier protein in, respectively, the degradation and synthesis pathways. These pathways are compartmentalized in the peroxisomes, mitochondria and cytosol of eukaryotic cells. Current structural knowledge of the enzymes comprising these pathways shows that the approximately 130 entries in the RCSB Protein Data Bank can be grouped into seven superfamilies. Multifunctional enzymes are important in both pathways.

Structure determination and refinement at 2.44 A resolution of argininosuccinate lyase from Escherichia coli.

Escherichia coli argininosuccinate lyase has been crystallized from a highly concentrated sample of purified recombinant alpha-methylacyl-CoA racemase, in which it occurred as a minor impurity. The structure has been solved using molecular replacement at 2.44 A resolution. The enzyme is tetrameric, but in this crystal form there is a dimer in the asymmetric unit. The tetramer has four active sites; each active site is constructed from loops of three different subunits. One of these catalytic loops, near residues Ser277 and Ser278, was disordered in previous structures of active lyases, but is very well ordered in this structure in one of the subunits owing to the presence of two phosphate ions in the active-site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex.

A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2.

2-Enoyl-CoA hydratase 2, a part from multifunctional enzyme type 2, hydrates trans-2-enoyl-CoA to 3-hydroxyacyl-CoA in the (3R)-hydroxy-dependent route of peroxisomal beta-oxidation of fatty acids. Unliganded and (3R)-hydroxydecanoyl coenzyme A-complexed crystal structures of 2-enoyl-CoA hydratase 2 from Candida tropicalis multifunctional enzyme type 2 were solved to 1.95- and 2.35-A resolution, respectively. 2-Enoyl-CoA hydratase 2 is a dimeric, alpha+beta protein with a novel quaternary structure. The overall structure of the two-domain subunit of eukaryotic 2-enoyl-CoA hydratase 2 resembles the homodimeric, hot dog fold structures of prokaryotic (R)-specific 2-enoyl-CoA hydratase and beta-hydroxydecanoyl thiol ester dehydrase. Importantly, though, the eukaryotic hydratase 2 has a complete hot dog fold only in its C-domain, whereas the N-domain lacks a long central alpha-helix, thus creating space for bulkier substrates in the binding pocket and explaining the observed difference in substrate preference between eukaryotic and prokaryotic enzymes. Although the N- and C-domains have an identity of <10% at the amino acid level, they share a 50% identity at the nucleotide level and fold similarly. We suggest that a subunit of 2-enoyl-CoA hydratase 2 has evolved via a gene duplication with the concomitant loss of one catalytic site. The hydrogen bonding network of the active site of 2-enoyl-CoA hydratase 2 resembles the active site geometry of mitochondrial (S)-specific 2-enoyl-CoA hydratase 1, although in a mirror image fashion. This arrangement allows the reaction to occur by similar mechanism, supported by mutagenesis and mechanistic studies, although via reciprocal stereochemistry.

Crystallization and preliminary crystallographic data of 2-enoyl-CoA hydratase 2 domain of Candida tropicalis peroxisomal multifunctional enzyme type 2.

In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal multifunctional enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.