The coronavirus pandemic has put on hold contract-extension talks between All-Star shortstop Javier Baez and the Chicago Cubs.

China’s National Development and Reform Commission (the NDRC) issued a Circular on Policies of Improving the Electricity Price for On-Grid Wind Power (the Circular) at the end of May 2019. According to the Circular, the price of electricity from offshore wind power projects is cut to 0.8 yuan per kWh [US $0.12 per kWh] in 2019 and will further drop to 0.75 yuan [US $0.11] per kWh in 2020.

Gas peaker plants may be among the first casualties of a new Minnesota law requiring utilities to include energy storage as part of their long-range plans.

This week, Canadian Solar said that it has won three solar power contracts with Alberta’s Ministry of Infrastructure, for a total of 94 megawatts (MWp) of solar power system in southeast Alberta, with an average contracted PPA price of 48.05 Canadian dollars [US 36.27] per MWh. When in operation in 2021, these solar plants will provide 55 percent of the electricity needs for Alberta provincial government.

As utility customers invest more in advanced energy technologies, buildings are becoming dynamic energy hubs. Anywhere, anytime, a building could be a consumer, storage point, or generator of energy. And a building’s energy profile could change instantaneously as it responds to signals from internal controls, the grid operator, or third parties to deploy assets like controllable water heaters, smart equipment, solar, and storage.

A significant step to develop the first megawatt from the marine hydrokinetic (MHK) Sotenas Wave Energy Plant off of Sweden’s west coast took place earlier this month when a 120-ton subsea generator switchgear was deployed and connected to the Swedish national power grid via a 10-km-long subsea cable.
 

Gas peaker plants may be among the first casualties of a new Minnesota law requiring utilities to include energy storage as part of their long-range plans.

Presentation by George Milad of Uyemura International Corporation

Last week India finally held its national solar auction, the first in two years, seen as the least risky of several national and state-level solar auctions held over the past few years.

China is mulling a policy to provide a subsidy for lithium batteries deployed in electric vehicles. This new favorable policy is expected to propel the development of the country’s electric vehicle sector. China has existing subsidy policies for the sector, but higher prices for the parts used in electric vehicles, in particular lithium batteries, prevent many consumers from purchasing the vehicles.

By Kelly Scanlon, director, environment, health and safety policy and research, IPC The U.S. Environmental Protection Agency (EPA) released “scoping documents” for its upcoming reviews of certain high-priority chemical substances under the Toxic Substances Control Act (TSCA) Section 6(b). Your review of these documents is important because scoping is the foundation of the risk evaluation […]

According to an analysis by the SUN DAY Campaign of data just released by the Federal Energy Regulatory Commission (FERC), natural gas dominated new electrical generating capacity in 2018. However, renewable energy sources (i.e., biomass, geothermal, hydropower, solar, wind) seem poised to swamp fossil fuels as new generating capacity is added over the next three years.

China’s National Development and Reform Commission (the NDRC) issued a Circular on Policies of Improving the Electricity Price for On-Grid Wind Power (the Circular) at the end of May 2019. According to the Circular, the price of electricity from offshore wind power projects is cut to 0.8 yuan per kWh [US $0.12 per kWh] in 2019 and will further drop to 0.75 yuan [US $0.11] per kWh in 2020.

Sicily's regional government is offering to subsidise holidays on the island for both domestic and international visitors in an effort to kickstart tourism after the coronavirus pandemic, the Guardian writes. Current plans include subsidising visitors' accommodation costs, as well as vouchers for cultural and heritage activities. It may also pay for up to half of the cost of flights, but this has not been confirmed yet.

Fossil fuels are reaping $550 billion a year in subsidies and holding back investment in cleaner forms of energy, the International Energy Agency said.

Eversheds Sutherland advises Glennmont on the sale of a minority stake in 211MW Finnish wind project Eversheds Sutherland has advised Glennmont Partners on its agreed sale of a 15% minority stake in Project Piiparinmäki, a 211.4MW wind farm, to...

Syria's oil and mineral resources ministry announced Saturday a reduction in automobile fuel subsidies, the latest government measure to tackle a deepening economic crisis.

The Premier League will warn clubs at a meeting on Monday, not to vote against the season restarting at neutral venues, as it could cost them more than relegation, according to Sky Sports. The clubs in the bottom six – Brighton, West Ham, Watford, Bournemouth, Aston Villa and Norwich – have reservations about proposals to […]

COVID-19: EPL threatens clubs over restarting matches at neutral venues

DStv has come under fire from subscribers demanding a decrease in their monthly instalments due to the lack of live sport for broadcast.

Covid-19 has tipped some struggling and startup businesses over already - but the real toll will emerge in the coming months. READ MORE:• Receiver says wage subsidy propping up a walking-dead army of 'zombie firms' John...

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SEOUL, South Korea (AP) — South Korea's capital has shut down more than 2,100 nightclubs, hostess bars and discos after dozens of coronavirus infections were linked to club goers who went out last weekend as the country relaxed social distancing guidelines. The measures imposed Saturday by Seoul Mayor Park Won-soon came after the national government urged entertainment venues around the nation to close or otherwise enforce anti-virus measures, including distancing, temperature checks, keeping customer lists and requiring employees to wear masks. Park said the entry bans on the facilities will be maintained until the...

The International Football Association Board (IFAB) confirmed on May 8 that teams can make extra substitutions during games to "protect player welfare" in response to the coronavirus pandemic.

Do you import or otherwise place products on the EEA market? If so, you should be aware of the new European Chemicals Agency (“ECHA”) SCIP database. Companies placing products (articles) on the EEA market, including companies importing s...

South Korean health authorities are investigating a small but growing coronavirus outbreak centered in a handful of Seoul nightclubs, seeking to keep infections in check ...

Teams face likely fixture congestion in a packed calendar as they attempt to make up for lost time when play can resume

Efforts to fast-track the opening of pubs next month have been delivered a body blow by Chief Medical Officer Dr Tony Holohan.

Irish golf courses will reopen on 18 May after 55 days without a round being played - but there will be numerous restrictions. Gary Moran reports on the sport's first, tentative steps towards normality.

South Korea confirmed 12 new coronavirus cases on Friday, its first increase above 10 in five days, as authorities warned numbers would increase as it had identified a new cluster of infections linked to a 29-year-old man.The IT company employee had spread the virus to at least 14 others as he wandered around the capital Seoul and four neighbouring cities over a long weekend period at the start of the month, said the Korea Centres for Disease Control and Prevention.While the 12 cases were those…

As Asia’s rich contemplate where to place their nest eggs, most have overwhelmingly chosen Singapore over rival international financial centre Hong Kong so far this year, according to the region’s largest wealth manager.High-net-worth individuals have mainly instructed their private bankers at UBS to place new money in Singapore rather than Hong Kong, said Edmund Koh, the Swiss bank’s president in the Asia-Pacific region. Last year, the region’s high-net-worth individuals opened more new…

The positions are for a non-renewable term of six years.

Tata Value Homes Limited (TVHL), a 100% subsidiary of Tata Housing Development Company Limited, today strengthened its presence in South India by developing Spanish themed residential township at Sriperumbudur, near Chennai. The company plans to Invest around Rs 300 crore in setting up this township. Inspired by Spanish living, Santorini is designed by architects F+A. The township is spread across lush greenery at Sriperumbudur, one of the fastest growing corridors in Chennai. Speaking on the project Brotin Banerjee, MD & CEO, Tata Housing Development Co. Ltd., said, with rapid urbanisation and influx of global lifestyle trends more and more affluent home buyers are looking for homes to reflect the financial […]

FIFA's decision to temporarily allow up to five substitutions per match to help cope with potential fixture congestion was met coolly in South America on Friday.

Title: Scientists Discover Substance That Causes Pain
Category: Health News
Created: 4/28/2010 2:10:00 PM
Last Editorial Review: 4/29/2010 12:00:00 AM

Title: Teen Impulsiveness Has Different Sources in ADHD, Substance Use
Category: Health News
Created: 4/29/2012 4:05:00 PM
Last Editorial Review: 4/30/2012 12:00:00 AM

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Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

ABSTRACT

The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an artA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the hvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the hvpssA and hvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination.

IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

ABSTRACT

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.

IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.

Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

ABSTRACT

Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.

IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.

ABSTRACT

Microbial pathogens exploit host nutrients to proliferate and cause disease. Intracellular pathogens, particularly those exclusively living in the phagosome such as Histoplasma capsulatum, must adapt and acquire nutrients within the nutrient-limited phagosomal environment. In this study, we investigated which host nutrients could be utilized by Histoplasma as carbon sources to proliferate within macrophages. Histoplasma yeasts can grow on hexoses and amino acids but not fatty acids as the carbon source in vitro. Transcriptional analysis and metabolism profiling showed that Histoplasma yeasts downregulate glycolysis and fatty acid utilization but upregulate gluconeogenesis within macrophages. Depletion of glycolysis or fatty acid utilization pathways does not prevent Histoplasma growth within macrophages or impair virulence in vivo. However, loss of function in Pck1, the enzyme catalyzing the first committed step of gluconeogenesis, impairs Histoplasma growth within macrophages and severely attenuates virulence in vivo, indicating that Histoplasma yeasts rely on catabolism of gluconeogenic substrates (e.g., amino acids) to proliferate within macrophages.

IMPORTANCE Histoplasma is a primary human fungal pathogen that survives and proliferates within host immune cells, particularly within the macrophage phagosome compartment. The phagosome compartment is a nutrient-limited environment, requiring Histoplasma yeasts to be able to assimilate available carbon sources within the phagosome to meet their nutritional needs. In this study, we showed that Histoplasma yeasts do not utilize fatty acids or hexoses for growth within macrophages. Instead, Histoplasma yeasts consume gluconeogenic substrates to proliferate in macrophages. These findings reveal the phagosome composition from a nutrient standpoint and highlight essential metabolic pathways that are required for a phagosomal pathogen to proliferate in this intracellular environment.

ABSTRACT

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca²⁺-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol—and concomitant with the proteomic changes—lanthanum (La³⁺) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy.

IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La³⁺) is mostly substrate specific and that La³⁺ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

BACKGROUND Pregnant patients from rural counties of Western North Carolina face additional barriers when accessing comprehensive perinatal substance use disorders care at Project CARA as compared to patients local to the program in Buncombe County. We hypothesized regional patients would be less engaged in care.

METHOD Using a retrospective cohort design, univariate analyses (², t-test; P < .05) compared patients' characteristics, engagement in care, and delivery outcomes. Engagement in care, the primary outcome, was operationalized as: attendance at expected, program-specific prenatal and postpartum visits, utilization of in-house counseling, community-based and/or inpatient substance use disorders treatment, and maternal urine drug screen at delivery negative for illicit substances.

RESULTS Regional patients (n = 324) were more likely than Buncombe County patients (n = 284) to have opioid [209 (64.5%) versus 162 (57.0%)] or amphetamine/methamphetamine use disorders (25 [7.7%] versus 13 [4.6%]), but less likely to have cannabis use (19 [5.9%] versus 38 [13.4%]; P = .009) and concurrent psychiatric disorders (214 [66.0%] versus 220 [77.5%]; P = .002). Engagement at postpartum visits was the significantly different outcome between patients (110/221 [49.8%] versus 146/226 [64.6%]; P = .002).

LIMITATIONS Outcomes were available for 66.8% of regional and 79.6% of Buncombe County patients of one program in one predominately white, non-Hispanic region of the state.

CONCLUSION Contrary to our hypothesis, regional and Buncombe County women engaged in prenatal care equally. However, a more formal transition into the postpartum period is needed, especially for regional women. A "hub-and-spokes" model that extends delivery of perinatal substance use disorders care into rural communities may be more effective for engagement retention.

Studies of the former NE England coalfield in Tyneside demonstrated that heat flow perturbations in boreholes were due to the entrainment and lateral dispersion of heat from deeper in the subsurface through flooded mine workings. This work assesses the influence of historical mining on geothermal observations across Greater Glasgow. The regional heat flow for Glasgow is 60 mW m^–2 and, after correction for palaeoclimate, is estimated as c. 80 mW m^–2. An example of reduced heat flow above mine workings is observed at Hallside (c. 10 km SE of Glasgow), where the heat flow through a 352 m deep borehole is c. 14 mW m^–2. Similarly, the heat flow across the 199 m deep GGC01 borehole in the Glasgow Geothermal Energy Research Field Site is c. 44 mW m^–2. The differences between these values and the expected regional heat flow suggest a significant component of horizontal heat flow into surrounding flooded mine workings. This deduction also influences the quantification of deeper geothermal resources, as extrapolation of the temperature gradient above mine workings would underestimate the temperature at depth. Future projects should consider the influence of historical mining on heat flow when temperature datasets such as these are used in the design of geothermal developments.

Supplementary material: Background information on the chronology of historical mining at each borehole location and a summary of groundwater flow in mine workings beneath Glasgow are available at https://doi.org/10.6084/m9.figshare.c.4681100

Thematic collection: This article is part of the ‘Early Career Research’ available at: https://www.lyellcollection.org/cc/SJG-early-career-research

BACKGROUND:

There is a dearth of evidence regarding the association of use of electronic cigarettes (e-cigarettes) with certain product characteristics and adolescent and young adult risk of unhealthy tobacco use patterns (eg, frequency of combustible cigarette smoking), which is needed to inform the regulation of e-cigarettes.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.