Scholarly Works, Biochemistry
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Browsing Scholarly Works, Biochemistry by Subject "0601 Biochemistry and Cell Biology"
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- Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin BiosynthesisValentino, Hannah; Sobrado, Pablo (American Chemical Society, 2021-09-28)N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in someStreptomycessp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higherkcat/KMvalue for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where thekredis rate-limiting, FzmM exhibits a very fastkred(50 ± 0.01 s-1at 4 °C) with NADPH. NADPH binds at aKDvalue of ∼400 μM, and hydride transfer occurs withpro-Rstereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than thekcat. This reaction is enhanced in the presence of Asp with a slightly fasterkoxthan thekcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.
- Flavin oxidation in flavin dependent N-monooxygenaseSobrado, Pablo; Robinson, Reeder; Klancher, Catherine; Rodriguez, Pedro (2019-01-02)
- Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivityShi, Hao; Munk, Alexander; Nielsen, Thomas S.; Daughtry, Morgan R.; Larsson, Louise; Li, Shize; Hoyer, Kasper F.; Geisler, Hannah W.; Sulek, Karolina; Kjobsted, Rasmus; Fisher, Taylor; Andersen, Marianne M.; Shen, Zhengxing; Hansen, Ulrik K.; England, Eric M.; Cheng, Zhiyong; Hojlund, Kurt; Wojtaszewski, Jorgen FP P.; Yang, Xiaoyong; Hulver, Matthew W.; Helm, Richard F.; Treebak, Jonas T.; Gerrard, David E. (Elsevier, 2018-05-01)Objective: Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. Methods: We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. Results: We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Conclusions: Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders.
- Structural Determinants of Flavin Dynamics in a Class B MonooxygenaseCampbell, Ashley C.; Robinson, Reeder; Mena-Aguilar, Didier; Sobrado, Pablo; Tanner, John J. (American Chemical Society, 2020-12-08)The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-l-ornithine and NADP+. Crystal structures were determined for M101A in the resting state and complexed with NADP+. The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A-NADP+ complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP+ in preparation for a new catalytic cycle.