Structural and Functional Studies of Biotin-Dependent Carboxylases

A persisting question in biology concerns the exceptional diversity of metabolic enzymes and how they respond to their ligands and dynamic environments with remarkable precision. In humans, the family of biotin-dependent carboxylases holds important roles in intermediary metabolism. Recent years have witnessed significant progress toward understanding these enzymes' roles in homeostatic regulation. However, due to a lack of structural information, their catalytic mechanisms, as well as the macromolecular consequences of their genetic mutations, are still not well understood. This dissertation describes the characterization of two biotin-dependent carboxylases that catalyze essential metabolic transformations in humans and bacteria, using X-ray crystallography to elucidate their structures and biochemical assays to verify their activities. We engineer a novel chimeric variant of propionyl-CoA carboxylase (PCC) and produce the first crystal structure of its 750-kDa α6β6 holoenzyme. This structure reveals the architecture of PCC's twelve catalytic domains and allows the mapping of its disease-associated gene mutations to predict their effects on enzyme stability and catalysis. We also identify and describe a new domain that is integral to maintaining inter-subunit contacts within PCC. Following this, we extend our studies to methylcrotonyl-CoA carboxylase (MCC), another 750-kDa α6β6 holoenzyme that differs from PCC primarily in its substrate preference. The crystal structure of MCC assumes a markedly different configuration from PCC despite the high sequence identity between the two. Theorizing that these enzymes may represent unique lineages in the evolution of the biotin-dependent carboxylases, we apply similar approaches to the study of a third biotin-dependent carboxylase. Our efforts have produced the first two holoenzyme structures of CoA-recognizing biotin-dependent carboxylases, and provide valuable insight for understanding the functions of these vital enzymes

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Structure and substrate selectivity of the 750-kDa α6β6 holoenzyme of geranyl-CoA carboxylase.

Geranyl-CoA carboxylase (GCC) is essential for the growth of Pseudomonas organisms with geranic acid as the sole carbon source. GCC has the same domain organization and shares strong sequence conservation with the related biotin-dependent carboxylases 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC). Here we report the crystal structure of the 750-kDa α6β6 holoenzyme of GCC, which is similar to MCC but strikingly different from PCC. The structures provide evidence in support of two distinct lineages of biotin-dependent acyl-CoA carboxylases, one carboxylating the α carbon of a saturated organic acid and the other carboxylating the γ carbon of an α-β unsaturated acid. Structural differences in the active site region of GCC and MCC explain their distinct substrate preferences. Especially, a glycine residue in GCC is replaced by phenylalanine in MCC, which blocks access by the larger geranyl-CoA substrate. Mutation of this residue in the two enzymes can change their substrate preferences

Cultural Norms as a Source of Law: The Example of Bottled Water

As a metaphor for the interaction of law and culture, bottled water is striking in its simplicity and clarity. Bottled water consumers form a surprisingly loyal subculture of beverage drinkers, united by the water truths and water myths that they embrace. More recently, an equally fervent subculture of bottled water protestors has begun to coalesce. Notably, the cultural norms associated with both supporters and detractors extend beyond mere hydration and encompass such fundamental and varied notions as health, taste, convenience, status, morality, anti-privatization, sustainability, and truth-telling. In contrast to the cultural story, the legal narrative is surprisingly sparse, overlooking an important opportunity to engage in a cultural-legal dialogue on the evolving norms of water use. This Article argues that the states\u27 laws of water allocation -- evolved over more than a century from the customs of water users -- are uniquely suited to stimulate this unrealized dialectic and to translate social values into law. In particular, the Article identifies four discrete principles of water law that are especially relevant to the discourse, including reasonable use, beneficial use, preferred uses, and the public interes

Spotlight on Poland: Negative Views of Russia Surge, but Ratings for U.S., NATO, EU Improve

The Russian invasion of Ukraine has led to a dramatic shift in attitudes in Poland, a key European partner and one which only three decades ago was part of the former Soviet Union's Eastern Bloc. Negative attitudes among Poles towards Russia are at all-time highs since Pew Research Center began tracking opinion on this question in 2007, with virtually unanimous negative opinions of the Russian state. Currently, 94% see Russia as a major threat, up from 65% who said this in 2018, and 94% have no confidence at all in Russian President Vladimir Putin -- also an all-time high. The sharp decline in positive attitudes toward Russia has benefited Poland's western allies, specifically the United States, the North Atlantic Treaty Organization (NATO) and the European Union (Poland is a member of both NATO and the EU). Around nine-in-ten Poles have a favorable view of the U.S., NATO and the EU, all of which represent the highest shares since 2007. In terms of Poland's relationship with the U.S., the increase in favorable attitudes toward America coincides with a strong 82% confidence rating for U.S. President Joe Biden, a marked increase from the 51% who had confidence in former President Donald Trump in 2019. In addition, roughly two-thirds in Poland see having a close relationship with the U.S. as more important than having one with Russia. Only 1% want a closer relationship with Russia, while 28% volunteer that both are equally important. Just three years ago, more than half of Poles (53%) offered that both relationships are equally important

Chinas Partnership With Russia Seen as Serious Problem for the U.S.

As war rages in Ukraine – one which China thus far has refused to condemn – Americans are acutely concerned about the partnership between China and Russia. Around nine-in-ten U.S. adults say it's at least a somewhat serious problem for the United States, and a 62% majority say it's a very serious problem – more than say the same about any of the other six problems asked about, including China's involvement in politics in the U.S., its policies on human rights and tensions between China and Taiwan, among others.

Cultural Norms as a Source of Law: The Example of Bottled Water

As a metaphor for the interaction of law and culture, bottled water is striking in its simplicity and clarity. Bottled water consumers form a surprisingly loyal subculture of beverage drinkers, united by the water truths and water myths that they embrace. More recently, an equally fervent subculture of bottled water protestors has begun to coalesce. Notably, the cultural norms associated with both supporters and detractors extend beyond mere hydration and encompass such fundamental and varied notions as health, taste, convenience, status, morality, anti-privatization, sustainability, and truth-telling. In contrast to the cultural story, the legal narrative is surprisingly sparse, overlooking an important opportunity to engage in a cultural-legal dialogue on the evolving norms of water use. This Article argues that the states\u27 laws of water allocation -- evolved over more than a century from the customs of water users -- are uniquely suited to stimulate this unrealized dialectic and to translate social values into law. In particular, the Article identifies four discrete principles of water law that are especially relevant to the discourse, including reasonable use, beneficial use, preferred uses, and the public interes

Is there life? Is there spirit? Debating belief and being a good Christian in Watchman Nee’s ‘Little Flock’ : Religions

Funding: This research was funded by the Shanghai Social Science Innovation Research Base, for ‘Research on Transitional Sociology with Chinese Characteristics’.Christian, especially Protestant, identity is often framed through the lens of belief, particularly belief understood as an interior orientation. Through an examination of the non-denominational Protestant group, the ‘Little Flock’, founded by Watchman Nee in the early 20th century, we trace enduring aspects of Little Flock theology in contemporary Chinese Protestant practice. In particular, we attend to conceptions of and debates surrounding belief and how to determine the quality of one’s faith—whether or not one might be considered not just a Christian, but a ‘good’ one.Publisher PDFPeer reviewe