Recruitment of a Middling Promiscuous Enzyme Drives Adaptive Metabolic Evolution in Escherichia coli.

A key step in metabolic pathway evolution is the recruitment of promiscuous enzymes to perform new functions. Despite the recognition that promiscuity is widespread in biology, factors dictating the preferential recruitment of one promiscuous enzyme over other candidates are unknown. Escherichia coli contains four sugar kinases that are candidates for recruitment when the native glucokinase machinery is deleted-allokinase (AlsK), manno(fructo)kinase (Mak), N-acetylmannosamine kinase (NanK), and N-acetylglucosamine kinase (NagK). The catalytic efficiencies of these enzymes are 103- to 105-fold lower than native glucokinases, ranging from 2,400 M-1 s-1 for the most active candidate, NagK, to 15 M-1 s-1 for the least active candidate, AlsK. To investigate the relationship between catalytic activities of promiscuous enzymes and their recruitment, we performed adaptive evolution of a glucokinase-deficient E. coli strain to restore glycolytic metabolism. We observed preferential recruitment of NanK via a trajectory involving early mutations that facilitate glucose uptake and amplify nanK transcription, followed by nonsynonymous substitutions in NanK that enhance the enzyme's promiscuous glucokinase activity. These substitutions reduced the native activity of NanK and reduced organismal fitness during growth on an N-acetylated carbon source, indicating that enzyme recruitment comes at a cost for growth on other substrates. Notably, the two most active candidates, NagK and Mak, were not recruited, suggesting that catalytic activity alone does not dictate evolutionary outcomes. The results highlight our lack of knowledge regarding biological drivers of enzyme recruitment and emphasize the need for a systems-wide approach to identify factors facilitating or constraining this important adaptive process.

Biochemical methods to map and quantify allosteric motions in human glucokinase.

Allosteric regulation of protein function is ubiquitous in biology. Allostery originates from ligand-mediated alterations in polypeptide structure and/or dynamics, which produce a cooperative kinetic or thermodynamic response to changing ligand concentrations. Establishing a mechanistic description of individual allosteric events requires both mapping the relevant changes in protein structure and quantifying the rates of differential conformational dynamics in the absence and presence of effectors. In this chapter, we describe three biochemical approaches to understand the dynamic and structural signatures of protein allostery using the well-established cooperative enzyme glucokinase as a case study. The combined application of pulsed proteolysis, biomolecular nuclear magnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry offers complementary information that can used to establish molecular models for allosteric proteins, especially when differential protein dynamics are involved.

Revisiting the manzamine biosynthetic hypothesis.

Covering: up to 2023The marine environment represents a rich yet challenging source of novel therapeutics. These challenges are best exemplified by the manzamine class of alkaloids, featuring potent bioactivities, difficult procurement, and a biosynthetic pathway that has eluded characterization for over three decades. This review highlights postulated biogenic pathways toward the manzamines, evaluated in terms of current biosynthetic knowledge and metabolic precedent.

Probing the 14-3-3 Isoform-Specificity Profile of Protein-Protein Interactions Stabilized by Fusicoccin A.

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein-protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. Recently, FC has emerged as an important chemical probe of human 14-3-3 PPIs involved in cancer and neurobiology. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of 14-3-3 isoforms on FC activity remains underexplored. This is a relevant question for the continued development of FC variants because there are seven isoforms of 14-3-3 in humans. Despite their sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions in vivo. Herein, we interrogate the isoform-specificity profile of FC in vitro using recombinant 14-3-3 isoforms and a library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal recognition domains of client proteins that are characterized targets of FC in vivo. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3σ. Together, these data support the feasibility of developing FC variants with enhanced isoform selectivity.

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease.

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.

Analysis of Interactions Stabilized by Fusicoccin A Reveals an Expanded Suite of Potential 14-3-3 Binding Partners.

Fusicoccin A (FC) is a diterpene glycoside that stabilizes protein-protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. Recently, FC has gained attention for its pro-apoptotic and neuroprotective properties in cell culture. Although the exact molecular mechanism(s) is (are) unresolved, 14-3-3 PPIs are central to this activity. With the goal of refining the pharmacology of this chemotype, we conducted a systematic analysis of the structural features that govern FC-induced stabilization of 14-3-3 PPIs utilizing a C-terminal phosphorylation recognition motif. This study confirmed that a C-terminal amino acid with a small alkyl group is required for the interaction of FC at canonical C-terminal 14-3-3 PPI interfaces. Using bioinformatics, this structural insight was leveraged to assemble a database of 119 candidate 14-3-3 PPIs that can serve as targets for FC. This group includes a subset of proteins with experimentally determined C-terminal phosphosites that have not been explored as potential targets of FC.

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation.

Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.

Molecular and cellular regulation of human glucokinase.

Glucose metabolism in humans is tightly controlled by the activity of glucokinase (GCK). GCK is predominantly produced in the pancreas, where it catalyzes the rate-limiting step of insulin secretion, and in the liver, where it participates in glycogen synthesis. A multitude of disease-causing mutations within the gck gene have been identified. Activating mutations manifest themselves in the clinic as congenital hyperinsulinism, while loss-of-function mutations produce several diabetic conditions. Indeed, pharmaceutical companies have shown great interest in developing GCK-associated treatments for diabetic patients. Due to its essential role in maintaining whole-body glucose homeostasis, GCK activity is extensively regulated at multiple levels. GCK possesses a unique ability to self-regulate its own activity via slow conformational dynamics, which allows for a cooperative response to glucose. GCK is also subject to a number of protein-protein interactions and post-translational modification events that produce a broad range of physiological consequences. While significant advances in our understanding of these individual regulatory mechanisms have been recently achieved, how these strategies are integrated and coordinated within the cell is less clear. This review serves to synthesize the relevant findings and offer insights into the connections between molecular and cellular control of GCK.

Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding.

Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78➔Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.

Short Total Synthesis of [15N5]-Cylindrospermopsins from 15NH4Cl Enables Precise Quantification of Freshwater Cyanobacterial Contamination.

Fresh water cyanobacterial algal blooms represent a major health risk because these organisms produce cylindrospermopsin, a toxic, structurally complex, zwitterionic uracil-guanidine alkaloid recognized by the EPA as a dangerous drinking water contaminant. At present, the ability to detect and quantify the presence of cylindrospermospin in water samples is severely hampered by the lack of an isotopically labeled standard for analytical mass spectrometry. Herein, we present a concise, scaled total synthesis of 15N cylindrospermosin from 15N ammonium chloride, which leverages a unique stereoselective intramolecular double conjugate addition step to assemble the tricyclic guanidine core. In addition to providing the first pure isotopically labeled probe for precise quantification of this potent biotoxin in fresh water sources, our results demonstrate how unique constraints associated with isotope incorporation compel novel solutions to synthesis design.

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism.

Human glucokinase (GCK) acts as the body's primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and ß-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a ß-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the ß-type variant lacks a burst phase. Comparative hydrogen-deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active site loop, which folds upon binding of glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a ß-strand located near the enzyme's hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, ß-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas ß-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK's unique kinetic cooperativity.

Between-User Reliability of Tier 1 Exposure Assessment Tools Used Under REACH.

When applying simple screening (Tier 1) tools to estimate exposure to chemicals in a given exposure situation under the Registration, Evaluation, Authorisation and restriction of CHemicals Regulation 2006 (REACH), users must select from several possible input parameters. Previous studies have suggested that results from exposure assessments using expert judgement and from the use of modelling tools can vary considerably between assessors. This study aimed to investigate the between-user reliability of Tier 1 tools. A remote-completion exercise and in person workshop were used to identify and evaluate tool parameters and factors such as user demographics that may be potentially associated with between-user variability. Participants (N = 146) generated dermal and inhalation exposure estimates (N = 4066) from specified workplace descriptions ('exposure situations') and Tier 1 tool combinations (N = 20). Interactions between users, tools, and situations were investigated and described. Systematic variation associated with individual users was minor compared with random between-user variation. Although variation was observed between choices made for the majority of input parameters, differing choices of Process Category ('PROC') code/activity descriptor and dustiness level impacted most on the resultant exposure estimates. Exposure estimates ranging over several orders of magnitude were generated for the same exposure situation by different tool users. Such unpredictable between-user variation will reduce consistency within REACH processes and could result in under-estimation or overestimation of exposure, risking worker ill-health or the implementation of unnecessary risk controls, respectively. Implementation of additional support and quality control systems for all tool users is needed to reduce between-assessor variation and so ensure both the protection of worker health and avoidance of unnecessary business risk management expenditure.

Mortality Among Hardmetal Production Workers: UK Cohort and Nested Case-Control Studies.

OBJECTIVE: The aim of this study was to characterize the mortality at two hardmetal production factories in the United Kingdom as part of an international study. METHODS: Standardized mortality ratios (SMRs) were calculated on the basis of mortality rates for England and Wales, and local rates. A nested case-control study of lung cancer was undertaken. RESULTS: The cohort comprised 1538 workers, with tracing complete for 94.4%. All-cause mortality was statistically significantly low for all cancers and nonmalignant respiratory disease, and for lung cancer was nonsignificantly low. The SMR for lung cancer for maintenance workers was elevated, based on only six deaths. The odds ratio for lung cancer per year of exposure to hardmetal was 0.93 (0.76 to 1.13). CONCLUSIONS: In this small study, there is no evidence to support that working in the UK hardmetal manufacturing industry increased mortality from any cause including lung cancer.

Antidiabetic Disruptors of the Glucokinase-Glucokinase Regulatory Protein Complex Reorganize a Coulombic Interface.

The glucokinase regulatory protein (GKRP) plays an essential role in glucose homeostasis by acting as a competitive inhibitor of glucokinase (GCK) and triggering its localization to the hepatocyte nucleus upon glucose deprivation. Metabolites such as fructose 6-phosphate and sorbitol 6-phosphate promote assembly of the GCK-GKRP complex, whereas fructose 1-phosphate and functionalized piperazines with potent in vivo antidiabetic activity disrupt the complex. Here, we establish the molecular basis by which these natural and synthetic ligands modulate the GCK-GKRP interaction. We demonstrate that a small-molecule disruptor of the protein-protein interaction utilizes a two-step conformational selection mechanism to associate with a rare GKRP conformation constituting 3% of the total population. Conformational heterogeneity of GKRP is localized to the N-terminus and deleting this region eliminates the ability of sorbitol 6-phosphate to promote the GCK-GKRP interaction. Stabilizing ligands favor an extended N-terminus, which sterically positions two arginine residues for optimal Coulombic interaction with a pair of carboxylate side chains from GCK. Conversely, disruptors promote a more compact N-terminus in which an interfacial arginine residue is stabilized in an unproductive orientation through a cation-π interaction with tyrosine 75. Eliminating the ability to sample this binding impaired conformation enhances the intrinsic inhibitory activity of GKRP. Elucidating the molecular basis of ligand-mediated control over the GCK-GKRP interaction is expected to impact the development and future refinement of therapeutic agents for diabetes and cardiovascular disease, which result from improper GKRP regulation of GCK.

Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic ß-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.

Kinetic Basis of Carbohydrate-Mediated Inhibition of Human Glucokinase by the Glucokinase Regulatory Protein.

The glucokinase regulatory protein (GKRP) is a competitive inhibitor of glucokinase (GCK), triggering its localization to the hepatocyte nucleus upon glucose deprivation. Here we establish the kinetic mechanism of GKRP action by analyzing its association with a genetically encoded, fluorescent variant of human GCK. Our results demonstrate that binding of GKRP to GCK involves two steps, formation of an initial encounter complex followed by conformational equilibration between two GKRP-GCK states. Fructose 6-phosphate, a known enhancer of GKRP action, promotes formation of the initial encounter complex via a 2.6-fold increase in kon and stabilizes the complex through a 60-fold decrease in koff.

Dual allosteric activation mechanisms in monomeric human glucokinase.

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In ß-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Kinetic Cooperativity in Human Pancreatic Glucokinase Originates from Millisecond Dynamics of the Small Domain.

The hallmark of glucokinase (GCK), which catalyzes the phosphorylation of glucose during glycolysis, is its kinetic cooperativity, whose understanding at atomic detail has remained open since its discovery over 40 years ago. Herein, by using kinetic CPMG NMR spectroscopic data for 17 isoleucine side chains distributed over all parts of GCK, we show that the origin of kinetic cooperativity is rooted in intramolecular protein dynamics. Residues of glucose-free GCK located in the small domain displayed distinct exchange behavior involving multiple conformers that are substantially populated (p>17 %) with a kex  value of 509±51 s(-1) , whereas in the glucose-bound form these exchange processes were quenched. This exchange behavior directly competes with the enzymatic turnover rate at physiological glucose concentrations, thereby generating the sigmoidal rate dependence that defines kinetic cooperativity.

Mortality of a cohort of workers in Great Britain with blood lead measurements.

OBJECTIVES: We examined the mortality of a historic cohort of workers in Great Britain with measured blood lead levels (BLLs). METHODS: SMRs were calculated with the population of Great Britain as the external comparator. Trends in mortality with mean and maximum BLLs and assessed lead exposure were examined using Cox regression. RESULTS: Mean follow-up length among the 9122 study participants was 29.2 years and 3466 deaths occurred. For all causes and all malignant neoplasms, the SMRs were statistically significantly raised. For disease groups of a priori interest, the SMR was significantly raised for lung cancer but not for stomach, brain, kidney, bladder or oesophageal cancers. The SMR was not increased for non-malignant kidney disease but was borderline significantly increased for circulatory diseases, for ischaemic heart disease (IHD) and cerebrovascular disease (CVD). No significant trends with exposure were observed for the cancers of interest, but for circulatory diseases and IHD, there was a statistically significant trend for increasing HR with mean and maximum BLLs. CONCLUSIONS: This study found an excess of lung cancer, although the risk was not clearly associated with increasing BLLs. It also found marginally significant excesses of IHD and CVD, the former being related to mean and maximum BLLs. The finding for IHD may have been due to lead, but could also have been due to other dust exposure associated with lead exposure and possibly tobacco smoking. Further work is required to clarify this and the carcinogenicity of lead.