Concatenation of engineered biocatalysts into multistep pathways dramatically increases their utility, but development of generalizable assembly methods remains a significant challenge. Herein we evaluate ‘bioretrosynthesis’, which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2,3-dideoxyinosine). Applying both directed evolution and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and 3-fold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase, but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.
To examine the impact of manual vs automated disc margin delineation on optic nerve head (ONH) and retinal nerve fiber layer (RNFL) parameters using spectral-domain optical coherence tomography (SDOCT).
A prospective cohort study consisting of normal, glaucoma suspect (GS) and glaucoma patients who underwent ONH and RNFL measurements using SDOCT technology (RTVue; Optovue Inc.). The retinal pigment epithelium/Bruch's membrane (RPE/BM) complex end points were automatically determined first, and were manually redefined subsequently. Analysis of variance, coefficient of variation (COV), concordance correlation coefficient (CCC), and Bland–Altman plots were used for the analyses.
Ninety-nine eyes of 50 subjects (age 68±10 years) consisting of 36 glaucoma, 56 GS, and 7 normal eyes were included. The RNFL thickness measurements were similar (P>0.05) between the two methods of demarcation, except for the inferior-nasal sector (P=0.04). For the ONH measurements, the cup-to-disc (C/D) ratio and rim area showed significant differences between the two methods (P<0.001). COV/CCC values for the ONH parameters were as follows: cup area 17.6%/0.88; cup volume 7.4%/0.91; average C/D ratio 18.1%/0.78; rim area 25.3%/0.69; and rim volume 42.6%/0.71, respectively. CCC/COV values for the RNFL parameters were as follows: average 2.1%/0.98; inferior-temporal quadrant 8.1%/0.79; inferior-nasal quadrant INQ quadrant 12.6%/0.67; SNQ quadrant 7.8%/0.83; and STQ quadrant 7.8%/0.88, respectively.
An overall high agreement and moderate–substantial concordance was observed between the demarcation methods. Automated disc margin delineation of SDOCT can be used reliably in clinical practice.
spectral-domain optical coherence tomography; glaucoma; retinal nerve fiber layer; optic nerve head; retinal pigment epithelium
Over a decade has passed since the elucidation of the first X-ray crystal structure of any complex II homolog. In the intervening time, the structures of five additional integral-membrane complex II enzymes and three homologs of the soluble domain have been determined. These structures have provided a framework for the analysis of enzymological studies of complex II superfamily enzymes, and have contributed to detailed proposals for reaction mechanisms at each of the two enzyme active sites, which catalyze dicarboxylate and quinone oxidoreduction, respectively. This review focuses on how structural data have augmented our understanding of catalysis by the superfamily.
Complex II; succinate dehydrogenase; succinate:quinone oxidoreductase; fumarate reductase; quinol:fumarate reductase; x-ray crystallography
Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PPi)-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PPi while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs.
Acetate kinase; PPi-dependent kinase; ASKHA superfamily
To examine the rate of macular thickness loss using time-domain optical coherence tomography (OCT) in functionally progressing versus non-progressing eyes, determined by standard automated perimetry (SAP).
Glaucoma suspects (GS) and glaucomatous (G) eyes underwent SAP and OCT imaging every 6 months. Functional progression was determined using pointwise linear regression, defined as 2 contiguous locations losing ≥1.0 dB/year at P<1.0% in the same hemifield. The annual rate of macular thickness loss was calculated from inner and outer regions of the macular map.
72 eyes (43 GS and 29G) with ≥30 months of follow-up were enroled. Fourteen eyes demonstrated SAP progression. The annual rate of macular thickness loss (μm/year) in progressing eyes was faster (all P<0.05) than non-progressing eyes in temporal outer (−1.90±2.97 vs 0.33±2.77), nasal inner (−1.70±2.66 vs 0.14±2.76), superior inner (−2.15±4.57 vs 0.51±2.99), temporal inner quadrants (−2.58±5.05 vs −0.38±2.34), and the average of inner macular quadrants (−1.84±2.90 vs 0.03±2.10). The rate of loss in the nasal inner (P=0.02) and temporal outer (P=0.02) macular regions was associated with optic disc haemorrhage.
Eyes with SAP progression have significantly greater rates of macular thickness loss consistent with glaucomatous retinal ganglion cell atrophy, as compared with non-progressing eyes.
glaucoma; macular thickness; imaging; progression
Prokaryotic phosphopentomutases (PPMs) are di-Mn2+ enzymes that catalyze the interconversion of α-d-ribose 5-phosphate and α-d-ribose 1-phosphate at an active site located between two independently-folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies on Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrate when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations on the PPMT85E phosphomimetic variant and the neutral corollary PPMT85Q identified that the side chain of Lys-240 changed conformation in response to active site charge, which modestly influenced affinity for the small molecule activator α-d-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated B. cereus PPM revealed a dramatic change in interdomain angle and a new hydrogen-bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen-bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to α-d-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPMD156A variant even under stringent activation conditions. These results permit a proposal for activation of PPM, and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase.
Phosphopentomutase; alkaline phosphatase superfamily; ribose 5-phosphate; ribose 1-phosphate; phosphoenzyme; catalytic nucleophile; metalloproteins; metalloenzymes
G protein-Coupled Receptors (GPCRs) use a complex series of intramolecular conformational changes to couple agonist binding to the binding and activation of cognate heterotrimeric G protein (Gαβγ). The mechanisms underlying this long-range activation have been identified using a variety of biochemical and structural approaches and have primarily used visual signal transduction via the GPCR rhodopsin and cognate heterotrimeric G protein transducin (Gt) as a model system. In this chapter, we will review the methods that have revealed allosteric signaling through rhodopsin and transducin. These methods can be applied to a variety of GPCR-mediated signaling pathways.
G protein coupled receptor; Heterotrimeric G proteins; Rhodopsin; Transducin; Receptor-mediated nucleotide exchange
Two crystal forms of an Mn2+-dependent phosphopentomutase were identified from chemically distinct conditions by sparse-matrix screening with and without the inclusion of 50 mM Mn2+. The crystals identified in the presence of Mn2+ were of dramatically better diffraction quality than those identified in the absence of added Mn2+.
Phosphopentomutases (PPMs) interconvert d-ribose 5-phosphate and α-d-ribose 1-phosphate to link glucose and nucleotide metabolism. PPM from Bacillus cereus was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Bacterial PPMs are predicted to contain a di-metal reaction center, but the catalytically relevant metal has not previously been identified. Sparse-matrix crystallization screening was performed in the presence or absence of 50 mM MnCl2. This strategy resulted in the formation of two crystal forms from two chemically distinct conditions. The crystals that formed with 50 mM MnCl2 were more easily manipulated and diffracted to higher resolution. These results suggest that even if the catalytically relevant metal is not known, the crystallization of putative metalloproteins may still benefit from supplementation of the crystallization screens with potential catalytic metals.
phosphopentomutase; alkaline phosphatase superfamily; ribose 5-phosphate, ribose 1-phosphate; sparse-matrix screening; metalloproteins; metalloenzymes
G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides or light. After activation, activated receptors interact with heterotrimeric G proteins and catalyze GDP release from the Gα subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the Gα protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (Gt), in phospholipid bilayers to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-Gt(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half life of functional complex to one week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes, and can extend the range of studies that investigate properties of these complexes.
Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitrosugar, l-evernitrose, analogs of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically-generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of 18O2 establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products, and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 Å resolution x-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-coA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.
Everninomicin; natural product biosynthesis; antibiotic biosynthesis; amine oxidation; nitrososynthase; flavin-containing monooxygenase; acyl-CoA dehydrogenase; induced fit
The carbohydrate-binding region of GspB from S. gordonii strain M99 was crystallized in space group P212121 and data were collected to 1.3 Å resolution.
The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 Å for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 Å for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall R
merge value of 0.04 and an R
merge value of 0.33 in the highest resolution shell.
GspB; glycoproteins; Streptococcus gordonii; sialic acid; adhesins; endocarditis; lectins
The carbohydrate binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspBBR) was expressed in Escherichia coli and purified using affinity and size exclusion chromatography. Separate sparse-matrix screening of GspBBR buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts, and additives supported crystallization of GspBBR in each buffer. While both sets of conditions supported crystal growth in space group P212121, these had distinct unit cell dimensions of a=33.3 Å, b=86.6 Å, c=117.9 Å for crystal form one and a=34.6 Å, b=98.3 Å, c=99.0 Å for crystal form two. Additive screening improved the crystals grown in both conditions such that diffraction extended beyond 2 Å resolution. A complete data set has been collected to 1.3 Å resolution with an overall Rsym value of 0.04 and an Rsym value of 0.33 in the highest resolution shell.
GspB; glycoprotein; Streptococcus gordonii; sialic acid; adhesin; endocarditis; lectin
GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspBBR), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspBBR structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspBBR-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.
The binding of bacteria to human platelets is thought to be important for development of infective endocarditis, a life-threatening infection of the cardiovascular system. Streptococcus gordonii is a leading cause of endocarditis. This pathogen uses a protein called GspB to attach to carbohydrates on human platelets. While this binding interaction appears to be mediated by a specific, contiguous domain within GspB, little is known about the molecular details of the interaction between GspB and the carbohydrate receptors on its human host. We therefore determined the crystal structure of the region of GspB that binds to platelet carbohydrates, both alone and in complex with a synthetic carbohydrate receptor. Using this structure as a guide, we were able to produce three strains of S. gordonii that lacked the ability to bind to platelet carbohydrates. One of these isogenic variants was studied more in-depth and lacked the ability to bind to human platelets in vitro and was reduced in virulence when tested in vivo. These studies provide the first structural information detailing the molecular interactions between any serine-rich repeat adhesin and its host receptor, and identify how different, related adhesins may have evolved different specificities for host receptors.
Phosphopentomutases (PPMs) interconvert d-ribose-5-phosphate and α-d-ribose-1-phosphate to link glucose and nucleotide metabolism. PPM from Bacillus cereus was overexpressed in Escherichia coli, purified to homogeneity, and crystallized. Bacterial PPMs are predicted to contain a di-metal reaction center, but the catalytically relevant metal had not previously been identified. Sparse matrix crystallization screening was performed in the presence or absence of 50 mM MnCl2. This strategy resulted in the formation of two crystal forms from two chemically distinct conditions. Crystals that formed with 50 mM MnCl2 were more easily manipulated and diffracted to higher resolution. These results suggest that even if the catalytically-relevant metal is not known, crystallization of putative metalloproteins may still benefit from supplementation of the crystallization screens with potential catalytic metals.
Phosphopentomutase; alkaline phosphatase superfamily; ribose-5-phosphate; ribose-1-phosphate; sparse matrix screening; metalloproteins; metalloenzymes
Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, with elevated intraocular pressure as an important risk factor. Increased resistance to outflow of aqueous humor through the trabecular meshwork causes elevated intraocular pressure, but the specific mechanisms are unknown. In this study, we used genome-wide SNP arrays to map the disease gene in a colony of Beagle dogs with inherited POAG to within a single 4 Mb locus on canine chromosome 20. The Beagle POAG locus is syntenic to a previously mapped human quantitative trait locus for intraocular pressure on human chromosome 19. Sequence capture and next-generation sequencing of the entire canine POAG locus revealed a total of 2,692 SNPs segregating with disease. Of the disease-segregating SNPs, 54 were within exons, 8 of which result in amino acid substitutions. The strongest candidate variant causes a glycine to arginine substitution in a highly conserved region of the metalloproteinase ADAMTS10. Western blotting revealed ADAMTS10 protein is preferentially expressed in the trabecular meshwork, supporting an effect of the variant specific to aqueous humor outflow. The Gly661Arg variant in ADAMTS10 found in the POAG Beagles suggests that altered processing of extracellular matrix and/or defects in microfibril structure or function may be involved in raising intraocular pressure, offering specific biochemical targets for future research and treatment strategies.
Primary open angle glaucoma (POAG) is a leading cause of vision loss and blindness affecting tens of millions of people. Ocular hypertension is a strong risk factor for the disease and the only effective target of treatment. Ocular hypertension results from increased resistance to outflow of aqueous humor through the trabecular meshwork, a specialized filtration tissue consisting of alternating layers of cells and connective tissue, but the specific reasons for the increased resistance are not known. The animal model for human POAG used in this study was a colony of Beagle dogs that carry an inherited form of the disease in which ocular hypertension is the primary manifestation. We have found a variant in ADAMTS10 that belongs to a family of genes that contribute to formation of extracellular matrix and may itself be involved in formation of elastic microfiber structures. We found that the ADAMTS10 protein is expressed at particularly high levels in the trabecular meshwork. The candidate variant in ADAMTS10 found in the POAG–affected Beagles suggests that altered processing of connective tissue and/or elastic microfiber defects may be involved in raising eye pressure, offering specific biochemical targets for future research and treatment strategies.
Experiments were conducted to compare the effects of 4-ethoxyamphetamine, a novel "designer" amphetamine, with (+)-amphetamine and an earlier "designer" amphetamine, 4-methoxyamphetamine, on rats. (+)-Amphetamine significantly decreased frequency threshold measures in an intracranial self-stimulation (ICSS) procedure using medial forebrain bundle electrodes, while 4-methoxyamphetamine and 4-ethoxyamphetamine increased these ICSS frequency thresholds. 4-Methoxyamphetamine and 4-ethoxyamphetamine had more potent effects on inhibition of uptake and stimulation of spontaneous release of 5-hydroxytryptamine (serotonin) than of dopamine. It is concluded that the neuropsychopharmacological profile of 4-ethoxyamphetamine is unlike that of (+)-amphetamine, but similar to that of 4-methoxyamphetamine, a potent hallucinogen in humans.
Rats were given single injections of vehicle or one of three doses of (+)-amphetamine (AM), 4-methoxyamphetamine (MA) or 4-ethoxyamphetamine (EA) after pretreatment with vehicle or reserpine, and vehicle or alpha-methyl-para-tyrosine (AMPT). EA is a "designer" drug that was recently seized from an illicit laboratory in Canada. Locomotion of the rats was recorded after treatment with the drugs, and whole brain levels of the drugs as well as monoamine neurotransmitters and their major acidic metabolites were then determined. Neither of the ring-substituted AM analogues influenced locomotion. AM induced locomotion in a dose-dependent manner, and this effect was blocked by AMPT but potentiated by reserpine. Brain concentrations of EA were lower than those of the other two drugs. The brain levels of monoamines and their metabolites indicate that AM releases a newly synthesized pool of dopamine which is transferred to vesicles after re-uptake. A very low dose of AM, but not higher doses, was found to elevate serotonin (5-hydroxytryptamine: 5-HT) levels independently of effects on catecholamines. Both MA and EA affected monoamine metabolites in a manner consistent with actions as reversible inhibitors of monoamine oxidase-an effect which has been previously demonstrated to be true for MA. Both drugs increased 5-HT levels at a very low dose, as did AM, but also increased noradrenaline levels at this dose. It is concluded that EA is not a psychomotor stimulant, but is similar in many of its effects to MA, a potent hallucinogen.