Structure and activity of the axon guidance protein MICAL (original) (raw)
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Febs Letters, 2000
Euglena gracilis, a protist having chloroplasts, has unique mitochondria, in which the common pyruvate and 2-oxoglutrate dehydrogenase multienzyme complexes are absent [1]. In the mitochondria, 2-oxoglutrate is converted to succinate through succinate semialdehyde by the sequential reactions of 2-oxoglutrate decarboxylase and succinate semialdehyde dehydrogenase. In contrast, pyruvate is oxidized in a CoAdependent reaction to form acetyl-CoA and CO 2 by the action of a single enzyme, pyruvate:NADP oxidoreductase (PNO) (EC 1.2.1.51), with NADP (but not NAD ) as an electron acceptor. It is suggested that these three enzymes participate in the respiratory metabolism of this organism. PNO, in contrast to the other two enzymes, has not yet been found in any other organisms. These unique characteristics of Euglena provide insight regarding the origin of mitochondria. In this communication, we propose that PNO was evolved from pyruvate:ferredoxin oxidoreductase (PFO) by linking with a £avoprotein with both FMN and FAD at the C-terminal side by gene fusion.
Free Radical Biology and Medicine, 1999
B-band lipopolysaccharide is an important virulence factor of the opportunistic pathogen Pseudomonas aeruginosa. WbpP is an enzyme essential for B-band lipopolysaccharide production in serotype O6. Sequence analysis suggests that it is involved in the formation of N-acetylgalacturonic acid. To test this hypothesis, overexpression and biochemical characterization of WbpP were performed. By using spectrophotometric assays and capillary electrophoresis, we show that WbpP is a UDP-GlcNAc C4 epimerase. The K m for UDP-GlcNAc and UDP-GalNAc are 197 and 224 M, respectively. At equilibrium, 70% of UDP-GalNAc is converted to UDP-GlcNAc, whereas the yield of the reverse reaction is only 30%. The enzyme can also catalyze the interconversion of non-acetylated substrates, although the efficiency of catalysis is significantly lower. Only 15 and 40% of UDP-Glc and UDP-Gal, respectively, are converted at equilibrium. WbpP contains tightly bound NAD(H) and does not require additional cofactors for activity. It exists as a dimer in its native state. This paper is the first report of expression and characterization of a C4 UDP-GlcNAc epimerase at the biochemical level. Moreover, the characterization of the enzymatic function of WbpP will help clarify ambiguous surface carbohydrate biosynthetic pathways in P. aeruginosa and other organisms where homologues of WbpP exist.
Scientific Reports, 2016
MICALs (Molecule Interacting with CasL) are conserved multidomain enzymes essential for cytoskeletal reorganization in nerve development, endocytosis, and apoptosis. In these enzymes, a type-2 calponin homology (CH) domain always follows an N-terminal monooxygenase (MO) domain. Although the CH domain is required for MICAL-1 cellular localization and actin-associated function, its contribution to the modulation of MICAL activity towards actin remains unclear. Here, we present the structure of a fragment of MICAL-1 containing the MO and the CH domains-determined by X-ray crystallography and small angle scattering-as well as kinetics experiments designed to probe the contribution of the CH domain to the actin-modification activity. Our results suggest that the CH domain, which is loosely connected to the MO domain by a flexible linker and is far away from the catalytic site, couples F-actin to the enhancement of redox activity of MICAL MO-CH by a cooperative mechanism involving a trans interaction between adjacently bound molecules. Binding cooperativity is also observed in other proteins regulating actin assembly/disassembly dynamics, such as ADF/Cofilins. Growing axons are guided to their appropriate targets by extracellular attractive or repulsive cues that are essential for proper neuronal growth and development, rewiring, fasciculation/defasciculation, and nerve regeneration after injury 1,2. Semaphorins, the most well characterized class of external repulsive guidance molecules, interact with Plexin and neuropilin receptors on axonal growth cones 3. Upon Plexin interaction with extracellular semaphorins, its cytosolic domain recruits and activates MICAL (Molecule Interacting with CasL); this activation promotes reorganization of the cytoskeleton and subsequent growth cone collapse 4,5. Since its initial identification in T-cells 6 , MICAL has also been found in a variety of neuronal and non-neuronal cell types in which it controls cytoskeletal dynamics 7,8. Three MICAL isoforms (MICAL-1,-2, and-3) have been identified in vertebrates 4,5. They have high overall sequence identity (1-2: 56%, 1-3: 56%, and the highest for 2-3: 65% in mouse MICALs). MICALs are large cytosolic proteins with an N-terminal flavoprotein monooxygenase (MO) domain containing an FAD cofactor followed by a variable number of protein-interaction domains 3. MICAL-1 combines the catalytic MO domain (residues 1-484) with three other domains thought to be important for modulating MICAL's activity and/or interaction with substrates: 1) a CH domain (residues 511-615), 2) a Lin-11 Isl-1 Mec-3 (LIM) domain (residues 666-761) 9 , and 3) a C-terminal region containing a coiled-coil Ezrin Radixin Moesin (ERM) domain 10,11. In addition, MICAL-1 contains a poly-proline PPKPP sequence (residues 830-834) that binds the SH3 domain of CasL 6. In mouse MICAL-1 (mMICAL-1, M W : 117 kDa, 1048 amino acids), the MO domain (MICAL MO) has been shown to reduce molecular oxygen to H 2 O 2 , with a ~70-fold preference for NADPH over NADH as the source of reducing equivalents 12. The structure of mMICAL-1 MO domain, determined by x-ray diffraction 12,13 , contains
Journal of Biological Chemistry 2013 Houben.pdf
The autotaxin ␣ splice variant (ATX␣) contains a unique polybasic insertion of unknown function. Results: ATX␣ binds strongly to heparin and cell-associated heparan sulfate. Conclusion: The ATX␣ insertion confers specific binding to heparan sulfate proteoglycans thereby targeting LPA production to the plasma membrane. Significance: ATX isoforms use distinct mechanisms to ensure spatially restricted LPA production and signaling.
2016
The redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding network of amicyanin from the aforementioned study were attempted to be used as a model to increase the stability of another beta barrel protein, the immunoglobulin light chain variable domain (V L). In addition, amicyanin was used as an alternative redox partner with MauG. MauG is a diheme protein from the mau gene cluster that catalyzes the biogenesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase (MADH). The amicyanin-MauG complex was used to study the free energy dependence and impact of reorganization energy in biological electron transfer reactions. The sole tryptophan of amicyanin was converted to a tyrosine via site-directed mutagenesis. This mutation had no effect on the electron transfer parameters with its redox partners, methylamine dehydrogenase and cytochrome c-551i. However, the pKa of the pH-dependence of the redox potential of the copper site was shifted +0.5 pH units. This was a result of an additional hydrogen bond between Met51 and the copper coordinating residue His95 in the reduced form of amicyanin. This additional hydrogen bond stabilizes the reduced form. Also, the stability of the copper site and the protein overall was significantly decreased, as seen by the temperature dependence of the visible spectrum of the copper site and the circular dichroism spectrum of the protein. This destabilization is attributed to the loss of an interior, crossbarrel hydrogen bond. The V L is structurally similar to amicyanin, but it does not contain any cross-barrel hydrogen bonds. The importance of the cross-barrel hydrogen bond in stabilizing amicyanin is evident. A homologous bond in iv V L was attempted to be engineered by using site-directed mutagenesis to insert neutral residues with protonatable groups into the core of the protein. Wild-type (WT) V L was purified from the periplasm and found to be properly folded as determined by circular dichroism and size exclusion chromatography. Mutants were expressed in E. coli using the amicyanin signal sequence for periplasmic expression. Folded mutant protein could not be purified from the periplasm. When amicyanin is used in complex with MauG, it retains the pH-dependence of the redox potential of its copper site due to the looseness of the interprotein interface. The free energy of the reaction was manipulated by variation in pH from pH 5.8 to 8.0. The ET parameters are reorganization energy of 2.34 eV and an electronic coupling constant of 0.6 cm-1. P94A amicyanin has a potential that is 120 mV higher than WT amicyanin and was used to extend the range of the free energy dependence studied. The ET parameters of the reaction of WT and P94A amicyanin with MauG were within error of each other. This is significant because the ET reaction of P94A amicyanin with its natural electron acceptor was not able to be studied due to a kinetic coupling of the ET step with a nonET step. This kinetic coupling obscured the parameters of the ET step because it is not kinetically distinguishable from the ET step. A Y294H MauG mutant was also studied. This mutation replaced the axial tyrosine ligand of the sixcoordinate heme of MauG with a histidine. No reaction is observed with Y294H MauG in its native reaction. However, the high valent oxidation state of the five-coordinate heme of Y294H MauG reacts with reduced amicyanin. The ET rate was analyzed by ET theory to study the high valent heme in Y294H MauG. The reorganization energy of Y294H MauG was calculated to be nearly 20% lower as compared to the same reaction with WT MauG. These results provide insight into the obscured nature of reorganization energy of large redox cofactors in proteins, particularly heme cofactors, as well as to how the active sites of enzymes are optimized to perform long range ET vs catalysis with regard to balancing redox potential and reorganization energy. v Dedicated to my family who has always supported me through every challenge Keishla, Danica Walt, Dianne, & Matt vi ACKNOWLEDGMENTS I express my deepest gratitude and appreciation to my famous, expert advisor and mentor, Dr. Victor L. Davidson for his guidance, patience, and example. In a world of fluorescent microscopes, he has been able to show me the utility and advantage of studying basic biochemistry and enzyme kinetics. Also I thank him for emphasizing the principle of Ockham's razor and not letting me (or anyone else in the lab) get swept away by overly complicated or unprecedented ideas. I thank my committee members, Suren Tatulian, William Self, and Kyle Rohde for technical support in performing experiments and also for supporting me and my work in the Burnett School of Biomedical Sciences. I also appreciate and thank Dr. Griff Parks, Dr. Sampath Parthasarathy, and Dr. Richard Peppler for their vision, guidance, and support of our lab and department as a whole. Particular thanks go to the rest of our lab group who have helped me keep my sanity, and, at times, tolerate my insanity. Specifically to Esha Sehanobish for her camaraderie, empathy, and teamwork; Heather Williamson for being a readily accessible font of biochemistry trivia; Yu Tang for her understanding and selfless willingness to express and purify proteins and provide buffers solutions, gels, and more. She is a most valuable anchor in the lab; Moonsung Choi and Sooim Shin for welcoming me to the lab and providing expertise in designing and performing experiments. Throughout my tenure at UCF, I have also enjoyed the company and friendship of many of my fellow students, especially Jason O. Matos, Aladdin Riad, and Richard Barrett, and everyone in the Biomedical Sciences Graduate Student Association. I thank them as well for helping me maintain my sanity. Finally, I thank my love, Keishla, and our beautiful daughter, Danica. They have always been on my mind or literally looking over my shoulder, demanding hard work, precision, integrity, and excellence. They have also both had unbelievable patience and understanding with me during my studies. Gracias. vii
Ayako Ohno et al., J. Mol. Biol (1998)
The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disul®de bridges. SMPI speci®cally inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The ®nal ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, C a , C H ) r.m.s.d. values of 0.45(AE0.11) A Ê and 0.57(AE0.18) A Ê for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two b-sheets, each consisting of four antiparallel b-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key b-barrel. One unique structural feature found in SMPI is in its extension between the ®rst and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain bg-crystallin proteins, because of its structural similarity to each domain of the bg-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the bg-crystallin superfamily are believed to have evolved.
Keresztessy et al 2006 Protein Science 15 2466-2480
Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the Nterminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q 6 , Q 8 , and Q 22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (K M app ¼ 250 mM, k cat ¼ 18.3 sec À1 , and k cat /K M app ¼ 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.
Structure of a Biological Oxygen Sensor: A New Mechanism for Heme-Driven Signal Transduction
Proceedings of The National Academy of Sciences, 1998
The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL's oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the ''on'' state without bound ligand and one in the ''off'' state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.
Drug metabolism and disposition: the biological fate of chemicals, 2011
Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N 1 -methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 Å. The crystals belong to space group P1, and two dimers are present in the unit cell. This work was supported by the Cluster of Excellence "Unifying Concepts in Catalysis" coordinated by the Technische Universitä t Berlin; and Fundaç ã o para a Ciê ncia e Tecnologia, Portugal [Grant SFRH/BD/37948/2007] (to C.C.) and Project [PTDC/QUI/64733/2006]. The exchange of researchers among laboratories involved in the work was funded by the Deutscher Akademischer Austauschdienst Programm-GRICES program.