Aspartate aminotransferase (AAT) is a prototypical pyridoxal 5′-phosphate (PLP) dependent enzyme that catalyzes the reversible interconversion of L-aspartate and α-ketoglutarate with oxalacetate and L-glutamate via a ping-pong catalytic cycle in which the pyridoxamine 5′-phosphate enzyme form is an intermediate. There is a bountiful literature on AAT that spans approximately 60 years, and much fundamental mechanistic information on PLP dependent reactions has been gained from its study. Here, we review our recent work on AAT, where we again used it as a test bed for fundamental concepts in PLP chemistry. First, we discuss the role that coenzyme protonation state plays in controlling reaction specificity, then ground state destabilization via hyperconjugation in the external aldimine intermediate is examined. The third topic is light enhancement of catalysis of Cα-H deprotonation by PLP in solution and in AAT, which occurs through a triplet state of the external aldimine intermediate. Lastly, we consider recent advances in our analyses of enzyme multiple sequence alignments for the purpose of predicting mutations that are required to interconvert structurally similar but catalytically distinct enzymes, and the application of our program JANUS to the conversion of AAT into tyrosine aminotransferase.
Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer.
Kinetic Isotope Effects; Marcus-like models; Nicotinamide; Folate; Hydrogen tunneling
This review describes the functions, structures, and mechanisms of nine nickel-containing enzymes: glyoxalase I, acireductone dioxygenase, urease, superoxide dismutase, [NiFe]-hydrogenase, carbon monoxide dehydrogenase, acetyl-coenzyme A synthase/decarbonylase, methyl-coenzyme M reductase, and lactate racemase. These enzymes catalyze their various chemistries by using metallocenters of diverse structures, including mononuclear nickel, dinuclear nickel, nickel-iron heterodinuclear sites, more complex nickel-containing clusters, and nickel-tetrapyrroles. Selected other enzymes are active with nickel, but the physiological relevance of this metal specificity is unclear. Additional nickel-containing proteins of undefined function have been identified.
Nickel; Enzyme; Metallocenter; Catalytic mechanism; Protein structure
MauG contains two c-type hemes with atypical physical and catalytic properties. While most c-type cytochromes function simply as electron transfer mediators, MauG catalyzes the completion of tryptophan tryptophylquinone (TTQ) biosynthesis within a precursor protein of methylamine dehydrogenase. This posttranslational modification is a six-electron oxidation that requires crosslinking of two Trp residues, oxygenation of a Trp residue and oxidation of the resulting quinol to TTQ. These reactions proceed via a bis-FeIV state in which one heme is present as FeIV=O and the other is FeIV with axial heme ligands provided by His and Tyr side chains. Catalysis does not involve direct contact between the protein substrate and either heme of MauG. Instead it is accomplished by remote catalysis using a hole hopping mechanism of electron transfer in which Trp residues of MauG are reversibly oxidized. In this process, long range electron transfer is coupled to the radical mediated chemical reactions that are required for TTQ biosynthesis.
Electron transfer; posttranslational modification; protein radical; peroxidase; oxygenase; high-valence iron
The flavoenzyme UDP-galactopyranose mutase (UGM) is a key enzyme in galactofuranose biosynthesis. The enzyme catalyzes the 6-to-5 ring contraction of UDP-galactopyranose to UDP-galactofuranose. Galactofuranose is absent in humans yet is an essential component of bacterial and fungal cell walls and a cell surface virulence factor in protozoan parasites. Thus, inhibition of galactofuranose biosynthesis is a valid strategy for developing new antimicrobials. UGM is an excellent target in this effort because the product of the UGM reaction represents the first appearance of galactofuranose in the biosynthetic pathway. The UGM reaction is redox neutral, which is atypical for flavoenzymes, motivating intense examination of the chemical mechanism and structural features that tune the flavin for its unique role in catalysis. These studies show that the flavin functions as nucleophile, forming a flavin-sugar adduct that facilitates galactose-ring opening and contraction. The 3-dimensional fold is novel and conserved among all UGMs, however the larger eukaryotic enzymes have additional secondary structure elements that lead to significant differences in quaternary structure, substrate conformation, and conformational flexibility. Here we present a comprehensive review of UGM three-dimensional structure, provide an update on recent developments in understanding the mechanism of the enzyme, and summarize computational studies of active site flexibility.
flavin-dependent reaction; galactofuranose; non-redox reaction; neglected diseases; tuberculosis; redox-switch; conformational changes; protein dynamics
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions of the murine Bax and human paraoxonase 1 (PON1) genes reportedly contain unique DRE-like sequences that respond to AhRs activated by some ligands but not others. Given the significant implications of this observation to understanding the diversity in AhR responses and that of other ligand-dependent nuclear receptors, a combination of DNA binding, nuclear translocation and gene expression analysis was used to investigate the molecular mechanisms underlying these ligand-selective responses. Although known AhR agonists stimulated AhR nuclear translocation, DRE binding and gene expression, the ligand-selective DRE-like DNA elements identified in the Bax and PON1 upstream regulatory regions failed to bind ligand-activated AhR or confer AhR-responsiveness upon a reporter gene. These results argue against the reported ligand-selectivity of AhR DNA binding and suggest DNA binding by ligand activated AhR involves DRE-containing DNA.
Aryl hydrocarbon receptor; Bax; Paraoxonase 1; DNA Binding
Conserved human cytochrome b5
(b5) residues D58 and D65 are critical for
interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for
stimulating the 17,20-lyase activity of CYP17A1. Here, we show that
b5 mutations E48G, E49G, D58G, and D65G have
reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone
6β-hydroxylation or nifedipine oxidation. The
b5 double mutation D58G/D65G fails to stimulate
these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G
retains 23–42% of wild-type stimulation. Neither mutation impairs the
activity stimulation of wild-type b5, nor does
mutation D58G/D65G impair the partial stimulation of mutations E48G or
E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl
serine afforded the highest testosterone 6β-hydroxylase activity with
wild-type b5 but the poorest activity with
b5 mutation E48G/E49G, and the activity
stimulation of mutation E48G/E49G was lost at [NaCl] > 50 mM.
Cross-linking of CYP3A4 and b5 decreased in the
order wild-type > E48G/E49G > D58G/D65G and varied with
phospholipid. We conclude that two b5 acidic
surfaces, primarily the domain including residues D58-D65, participate in the
stimulation of CYP3A4 activities. Our data suggest that a minor population of
CYP3A4 molecules remains sensitive to b5 mutation
E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity
Cytochrome b5; testosterone; CYP3A4; allostery; cytochrome P450; drug oxidation
Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.
cysteine oxidation; disulfide; hypothiocyanous acid; tubulin; hypochlorous acid
Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler Syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: PORnull, PORwt, PORYH, and PORVE, for which equivalent CYP1A2 and CYPOR levels were confirmed, except for PORnull, not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in PORYH and PORVE models than in PORwt, indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.
NADPH-cytochrome P450 oxidoreductase; Antley-Bixler Syndrome; POR; Polymorphism; Cytochrome P450; CYP1A2; P450 1A2; Protein-protein interaction; Drug-metabolizing enzymes; Adverse drug reactions
•Summary of key concepts in protein folding from the study of water-soluble proteins.•Discussion of the complexity of studying folding of outer membrane proteins (OMPs).•Role of periplasmic chaperones and the BAM complex in the folding of OMPs in vivo.•Examples of the application of biophysical methods to study OMP folding in vitro.•Comparisons of the folding of water-soluble proteins and OMPs.
Research into the mechanisms by which proteins fold into their native structures has been on-going since the work of Anfinsen in the 1960s. Since that time, the folding mechanisms of small, water-soluble proteins have been well characterised. By contrast, progress in understanding the biogenesis and folding mechanisms of integral membrane proteins has lagged significantly because of the need to create a membrane mimetic environment for folding studies in vitro and the difficulties in finding suitable conditions in which reversible folding can be achieved. Improved knowledge of the factors that promote membrane protein folding and disfavour aggregation now allows studies of folding into lipid bilayers in vitro to be performed. Consequently, mechanistic details and structural information about membrane protein folding are now emerging at an ever increasing pace. Using the panoply of methods developed for studies of the folding of water-soluble proteins. This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information. The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins.
Protein folding; Outer membrane protein; Periplasmic chaperone; BAM complex; Φ-Value analysis; Protein stability
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD+, then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD+ on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.
glyceraldehyde 3-phosphate dehydrogenase; glycolysis; enzyme kinetics; kinetic isotope effects; tuberculosis
The effect of tumor necrosis factor-α (TNFα) on cartilage matrix degradation is mediated by its transport and binding within the extracellular matrix (ECM) of the tissue, which mediates availability to cell receptors. Since the bioactive form of TNFα is a homotrimer of monomeric subunits, conversion between trimeric and monomeric forms during intratissue transport may affect binding to ECM and, thereby, bioactivity within cartilage. We studied the transport and binding of TNFα in cartilage, considering the quaternary structure of this cytokine. Competitive binding assays showed significant binding of TNFα in cartilage tissue, leading to an enhanced uptake. However, studies in which TNFα was cross-linked to remain in the trimeric form revealed that the binding of trimeric TNFα was negligible. Thus, binding of TNFα to ECM was associated with the monomeric form. Binding of TNFα was not disrupted by pre-treating cartilage tissue with trypsin, which removes proteoglycans and glycoproteins but leaves the collagen network intact. Therefore, proteoglycan loss during osteoarthritis should only alter the passive diffusion of TNFα but not its binding interaction with the remaining matrix. Our results suggest that matrix binding and trimer-monomer conversion of TNFα both play crucial roles in regulating the accessibility of bioactive TNFα within cartilage.
cartilage; TNFα; cytokine transport; binding; osteoarthritis; post traumatic osteoarthritis
Vitamin A (vitA) regulates obesity, insulin resistance, inflammation, dyslipidemia, and hemostasis through its metabolites retinaldehyde (Rald) and retinoic acid (RA) produced in endogenous enzymatic reactions. Combination of at least 3 of these conditions leads to development of metabolic syndrome (Msyn) and, consequently, type 2 diabetes and/or cardiovascular disease. Although many foods are fortified with vitA, it remains unknown what conditions of Msyn are influenced by moderate dietary vitA supplementation. A family of aldehyde dehydrogenase 1 (Aldh1) enzymes is a key contributor to obesity via sex- and fat depot-specific production of RA in adipose tissue. Therefore, we studied effects of moderate vitamin A supplementation of an obesogenic high-fat (HF) diet (4IU vitA/g and 20IU vitA/g HF diet) on multiple conditions and mediators of Msyn in wild-type (WT, C57Bl/6) and Aldh1a1−/− mice. We found that mild vitamin A supplementation did not influence obesity, fat distribution, and glucose tolerance in males and females of the same genotype. In contrast, multiplex analysis of bioactive proteins in blood showed moderately increased concentrations (10-15%) of inflammatory IL-18 and MIP-1γ in vitA supplemented vs. control WT males. Marked decrease (28-31%) in concentrations of lymphotactin and tissue factor, a key protein contributing to thrombogenesis during injury, was achieved by vitA supplementation in WT females compared to control WT females. Aldh1a1 deficiency reduced obesity, insulin resistance, suppressed many pro-inflammatory cytokines, and abolished the effects of vitA supplementation seen in WT mice. Our study revealed specific inflammatory and pro-thrombotic proteins in plasma regulated by dietary vitamin A and the critical role of endogenous vitA metabolism in these processes. The sex-specific decrease of plasma tissue factor concentrations by moderate dietary vitA supplementation could potentially reduce related pro-thrombotic states in obese females.
Retinol; coagulation; sex differences; Raldh1; HOMA-IR index
The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.
lipofuscin; A2E; retinoids; mass spectrometry; imaging; human; mouse; MALDI; profiling; RPE
Uptake, transport, and stabilization of xanthophylls in the human retina are important components of a complex multistep process that culminates in a nonuniform distribution of these important nutrients in the retina. The process is far from understood; here, we consider the potential role of interphotoreceptor retinoid-binding protein (IRBP) in this process. IRBP is thought to facilitate the exchange of 11-cis-retinal, 11-cis-retinol, and all-trans-retinol between the retinal pigment epithelium (RPE), photoreceptors and Müller cells in the visual cycle. Structural and biochemical studies suggest that IRBP has a variety of nonequivalent ligand binding sites that function in this process. IRBP is multifunctional, being able to bind a variety of physiologically significant molecules including fatty acids in the subretinal space. This wide range of binding activities is of particular interest because it is unknown whether the lutein and zeaxanthin found in the macula originate from the choroidal or retinal circulations. If from the choroidal circulation, then IRBP is a likely mediator for their transport across the interphotoreceptor matrix. In this report, we explore the binding interactions of retinoids, fatty acids, and carotenoids with IRBP using surface plasmon resonance (SPR)-based biosensors. IRBP showed similar affinity toward retinoids and carotenoids (1–2 µM), while fatty acids had approximately 10 times less affinity. These results suggest that further studies should be carried out to evaluate whether IRBP has a physiologically relevant role in binding lutein and zeaxanthin in the interphotoreceptor matrix.
Carotenoids; Interphotoreceptor matrix; Interphotoreceptor retinoidbinding protein; Macular pigments; Retinoids; SPR
Carotenoids and their metabolic derivatives serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, photoprotection and photosynthesis as well as cell signaling. These organic compounds are also important for visual function in vertebrate and non-vertebrate organisms. Enzymatic transformations of carotenoids to various apocarotenoid products are catalyzed by a family of evolutionarily conserved, non-heme iron-containing enzymes named carotenoid cleavage oxygenases (CCOs). Studies have revealed that CCOs are critically involved in carotenoid homeostasis and essential for the health of organisms including humans. These enzymes typically display a high degree of regio- and stereo-selectivity, acting on specific positions of the polyene backbone located in their substrates. By oxidatively cleaving or isomerizing specific double bonds, CCOs generate a variety of apocarotenoid isomer products. Recent structural studies have helped illuminate the mechanisms by which CCOs mobilize their lipophilic substrates from biological membranes to perform their characteristic double bond cleavage and/or isomerization reactions. In this review, we aim to integrate structural and biochemical information about CCOs to provide insights into their catalytic mechanisms.
carotenoid oxygenase; carotenoid; apocarotenoid; ACO; VP14; RPE65
Resonance Raman Spectroscopy (RRS) is a non-invasive method that has been developed to assess carotenoid status in human tissues including human skin in vivo. Skin carotenoid status has been suggested as a promising biomarker for human studies. This manuscript describes research done relevant to the development of this biomarker, including its reproducibility, validity, feasibility for use in field settings, and factors that affect the biomarker such as diet, smoking, and adiposity. Recent studies have evaluated the response of the biomarker to controlled carotenoid interventions, both supplement-based and dietary [e.g., provision of a high-carotenoid fruit and vegetable (F/V)-enriched diet], demonstrating consistent response to intervention. The totality of evidence supports the use of skin carotenoid status as an objective biomarker of F/V intake, although in the cross-sectional setting, diet explains only some of the variation in this biomarker. However, this limitation is also a strength in that skin carotenoids may effectively serve as an integrated biomarker of health, with higher status reflecting greater F/V intake, lack of smoking, and lack of adiposity. Thus, this biomarker holds promise as both a health biomarker and an objective indicator of F/V intake, supporting its further development and utilization for medical and public health purposes.
carotenoids; skin; resonance Raman spectroscopy; beta-carotene; biomarker
We investigated the effect of β-carotene (bC) supplementation during pregnancy in a mouse model of severe vitamin A deficiency, i.e. Lrat−/−Rbp−/− dams maintained on a vitamin A-deficient diet during gestation. bC, a provitamin A carotenoid, can be enzymatically cleaved to form vitamin A for use by the developing embryo. We found that an acute supplementation (13.5 days post coitum, dpc) of bC to Lrat−/−Rbp−/− dams on a vitamin A-deficient diet activated transcriptional mechanisms in the developing tissues to maximize the utilization of bC provided to the dams. Nevertheless, these regulatory mechanisms are inefficient under this regimen, as the embryonic phenotype was not improved. We further investigated the effect of a repeated supplementation of bC during a crucial developmental period (6.5–9.5 dpc) on the above-mentioned mouse model. This treatment improved the embryonic abnormalities, as 40% of the embryos showed a normal phenotype. In addition, analysis of retinoic acid-responsive genes, such as Cyp26a1 in these embryos suggests that bC cleavage results in the production of retinoic acid which then can be used by the embryo. Taken together, these in vivo studies show that bC can be used as a source of vitamin A for severely vitamin A-deficient mammalian embryos.
placenta; embryo; β-carotene; vitamin A deficiency; Lrat−/−Rbp−/−; retinoids
Intake of lycopene, a red, tetraterpene carotenoid found in tomatoes is epidemiologically associated with a decreased risk of chronic disease processes, and lycopene has demonstrated bioactivity in numerous in vitro and animal models. However, our understanding of absorption, tissue distribution, and biological impact in humans remains very limited. Lycopene absorption is strongly impacted by dietary composition, especially the amount of fat. Concentrations of circulating lycopene in lipoproteins may be further influenced by a number of variations in genes related to lipid absorption and metabolism. Lycopene is not uniformly distributed among tissues, with adipose, liver, and blood being the major body pools, while the testes, adrenals, and liver have the greatest concentrations compared to other organs. Tissue concentrations of lycopene are likely dictated by expression of and genetic variation in lipoprotein receptors, cholesterol transporters, and carotenoid metabolizing enzymes, thus impacting lycopene accumulation at target sites of action. The novel application of genetic evaluation in concert with lycopene tracers will allow determination of which genes and polymorphisms define individual lycopene metabolic phenotypes, response to dietary variables, and ultimately determine biological and clinical outcomes. A better understanding of the relationship between diet, genetics, and lycopene distribution will provide necessary information to interpret epidemiological findings more accurately and to design effective, personalized clinical nutritional interventions addressing hypotheses regarding health outcomes.
Lycopene; pharmacokinetics; biodistribution; bioavailability; genetics; tissue accumulation
The formal first step in in vitamin A metabolism is the conversion of its natural precursor β,β-carotene (C40) to retinaldehyde (C20) This reaction is catalyzed by the enzyme β,β-carotene-15,15′-monooxygenase (BCMO1). BCMO1 has been cloned from several vertebrate species, including humans. However, knowledge about this protein’s enzymatic and structural properties is scant. Here we expressed human BCMO1 in Spodoptera frugiperda 9 insect cells. Recombinant BCMO1 is a soluble protein that displayed Michaelis-Menten kinetics with a KM of 14 μM for β,β-carotene. Though addition of detergents failed to increase BCMO1 enzymatic activity, short chain aliphatic detergents such as C8E4 and C8E6 decreased enzymatic activity probably by interacting with the substrate binding site. Thus we purified BCMO1 in the absence of detergent. Purified BCMO1 was a monomeric enzymatically active soluble protein that did not require cofactors and displayed a turnover rate of about 8 molecules of β,β-carotene per second. The aqueous solubility of BCMO1 was confirmed in mouse liver and mammalian cells. Establishment of a protocol that yields highly active homogenous BCMO1 is an important step towards clarifying the lipophilic substrate interaction, reaction mechanism and structure of this vitamin A forming enzyme.
β,β-carotene; all-trans-retinal; vitamin A; β,β-carotene-15; 15′-monooxygenase; cytosolic; symmetric carotenoid cleavage; non-heme iron oxygenase
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-L-methionine (AdoMet) is converted to S-adenosyl-L-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the logV pH-profile was not well defined, plots of logV/K versus pH for AdoMet and phenylethanolamine, as well as the pKi versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.
mechanism; inhibition; ordered sequential; epinephrine; adrenaline; AdoMet
The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.
Isochorismate synthase; salicylate synthase; siderophore biosynthesis; enzyme engineering
Many therapeutic targets are cell surface receptors, which can be challenging antigens for antibody generation. For many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but also are internalized into the cell. This allows use of the antibody to deliver various payloads into the cell to achieve a therapeutic effect. Phage antibody technology has proven a powerful tool for the generation and optimization of human antibodies to any antigen. While applied to the generation of antibodies to purified proteins, it is possible to directly select cell binding and internalizing antibodies on cells. Potential advantages of this approach include: cell surface receptors are in native conformation on intact cells while this might not be so for recombinant proteins; antibodies can be selected for both cell binding and internalization properties; the antibodies can be used to identify their tumor associated antigens; and such antibodies can be used for human treatment directly since they are human in sequence.
This review will discuss the factors that impact the successful selection of cell binding and internalizing antibodies. These factors include the cell types used for selection, the impact of different phage antibody library formats, and the specific selection protocols used.
Phage antibody; antibody internalization; targeted drug delivery; cell selection; flow cytometry
Leishmaniasis is a vector-borne, neglected tropical disease caused by parasites from the genus Leishmania. Galactofuranose (Galf) is found on the cell surface of Leishmania parasites and is important for virulence. The flavoenzyme that catalyzes the isomerization of UDP-galactopyranose to UDP-Galf, UDP-galactopyranose mutase (UGM), is a validated drug target in protozoan parasites. UGMs from L. mexicana and L. infantum were recombinantly expressed, purified, and characterized. The isolated enzymes contained tightly bound flavin cofactor and were active only in the reduced form. NADPH is the preferred redox partner for both enzymes. A kcat value of 6 ± 0.4 s−1 and a Km value of 252 ± 42 μM were determined for L. infantum UGM. For L. mexicana UGM, these values were ~ 4-times lower. Binding of UDP-Galp is enhanced 10–20 fold in the reduced form of the enzymes. Changes in the spectra of the reduced flavin upon interaction with the substrate are consistent with formation of a flavin-iminium ion intermediate.
flavin-dependent reaction; galactofuranose; non-redox reaction; neglected diseases