Jodie E. Guy

Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes.

Desaturases and related enzymes perform O2-dependent dehydrogenations initiated at unactivated C-H groups with the use of a diiron active site. Determination of the long-sought oxidized desaturase crystal structure facilitated structural comparison of the active sites of disparate diiron enzymes. Experiments on the castor desaturase are discussed that provide experimental support for a hypothesized ancestral oxidase enzyme in the context of the evolution of the diiron enzyme diverse functionality. We also summarize recent analysis of a castor mutant desaturase that provides valuable insights into the relationship of proposed substrate-binding modes with respect to a range of catalytic outcomes.

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.

The crystal structure of the ivy Delta4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity.

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the Δ4 desaturation of 16:0-ACP and the Δ9 desaturation of 18:0-ACP and further desaturates Δ9-16:1 or Δ9-18:1 to the corresponding Δ4,9 dienes. The crystal structure of the enzyme has been solved to 1.95Å resolution, and both the iron-iron distance of ∼3.2Å and the presence of a μ-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His227 coordination to Fe2 is lost, and the side chain of Glu224, which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.

New Insights Into Multiple Coagulation Factor Deficiency from the Solution Structure of Human Mcfd2.

Human MCFD2 (multiple coagulation factor deficiency 2) is a 16-kDa protein known to participate in transport of the glycosylated human coagulation factors V and VIII along the secretory pathway. Mutations in MCFD2 or in its binding partner, the membrane-bound transporter ERGIC (endoplasmic reticulum-Golgi intermediate compartment)-53, cause a mild form of inherited hemophilia known as combined deficiency of factors V and VIII (F5F8D). While ERGIC-53 is known to be a lectin-type mannose binding protein, the role of MCFD2 in the secretory pathway is comparatively unclear. MCFD2 has been shown to bind both ERGIC-53 and the blood coagulation factors, but little is known about the binding sites or the true function of the protein. In order to facilitate understanding of the function of MCFD2 and the mechanism by which mutations in the protein cause F5F8D, we have determined the structure of human MCFD2 in solution by NMR. Our results show the folding of MCFD2 to be dependent on availability of calcium ions. The protein, which is disordered in the apo state, folds upon binding of Ca(2+) to the two EF-hand motifs of its C-terminus, while retaining some localized disorder in the N-terminus. NMR studies on two disease-causing mutant variants of MCFD2 show both to be predominantly disordered, even in the presence of calcium ions. These results provide an explanation for the previously observed calcium dependence of the MCFD2-ERGIC-53 interaction and, furthermore, clarify the means by which mutations in this protein result in inefficient secretion of blood coagulation factors V and VIII.

A single mutation in the castor Δ9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by ≈2 × 103-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

Crystal structure of the LMAN1‐CRD/MCFD2 transport receptor complex provides insight into combined deficiency of factor V and factor VIII

MINT‐7557086: lman1 (uniprotkb:P49257) and mcfd2 (uniprotkb:Q8NI22) bind (MI:0407) by X‐ray crystallography (MI:0114)

Crystal Structure of a Class XIB Phospholipase A2 (PLA2) RICE (ORYZA SATIVA) ISOFORM-2 PLA2 AND AN OCTANOATE COMPLEX

Phospholipase A2 catalyzes the specific hydrolysis of the sn-2 acyl bond of various glycerophospholipids, producing fatty acids and lysophospholipids. Phospholipase A2s (PLA2s) constitute a large superfamily of enzymes whose products are important for a multitude of signal transduction processes, lipid mediator release, lipid metabolism, development, plant stress responses, and host defense. The crystal structure of rice (Oryza sativa) isoform 2 phospholipase A2 has been determined to 2.0 Å resolution using sulfur SAD phasing, and shows that the class XIb phospholipases have a unique structure compared with other secreted PLA2s. The N-terminal half of the chain contains mainly loop structure, including the conserved Ca2+-binding loop, but starts with a short 310-helix and also includes two short anti-parallel β-strands. The C-terminal half is folded into three anti-parallel α-helices, of which the two first are also present in other secreted PLA2s and contain the conserved catalytic histidine and calcium liganding aspartate residues. The structure is stabilized by six disulfide bonds. The water structure around the calcium ion binding site suggests the involvement of a second water molecule in the mechanism for hydrolysis, the water-assisted calcium-coordinate oxyanion mechanism. The octanoate molecule in the complex structure is bound in a hydrophobic pocket, which extends to the likely membrane interface and is proposed to model the binding of the product fatty acid. Due to the differences in structure, the suggested surface for binding to the membrane has a different morphology in the rice PLA2 compared with other phospholipases.

The Crystal Structure of the Human Co-Chaperone P58IPK

P58IPK is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.

Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase

Heterodimers of a wild-type soluble lipid desaturase and a substrate-binding mutant demonstrate a reactivity that contrasts with membrane desaturases which require two active subunits. Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity.

The Crystal Structure of the Lumenal Domain of Erv41p, a Protein Involved in Transport between the Endoplasmic Reticulum and Golgi Apparatus

Erv41p is a conserved integral membrane protein that is known to play a role in transport between the endoplasmic reticulum and Golgi apparatus, part of the early secretory pathway of eukaryotes. However, the exact function of the protein is not known, and it shares very low sequence identity with proteins of known structure or function. Here we present the structure of the full lumenal domain of Erv41p from Saccharomyces cerevisiae, determined by X-ray crystallography to a resolution of 2.0Å. The structure reveals the protein to be composed predominantly of two large β-sheets that form a twisted β-sandwich. Comparison to structures in the Protein Data Bank shows that the Erv41p lumenal domain displays only limited similarity to β-sandwich domains of other proteins. Analysis of the surface properties of the protein identifies an extensive patch of negative electrostatic potential on the exposed surface of one of the β-sheets, which likely forms a binding site for a ligand or interaction partner. A predominantly hydrophobic region close to the membrane interface is identified as a likely site for protein-protein interaction. This structure, the first of Erv41p or any of its homologues, provides a new starting point for studies of the roles of Erv41p and its interaction partners in the eukaryotic secretory pathway.