Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66378/1/j.1432-1033.1991.tb16100.x.pd

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution

Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover

Identification and Properties of New Flavins in Electron-Transferring Flavoprotein from Peptostreptococcus elsdenii and Pig-Liver Glycolate Oxidase

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65168/1/j.1432-1033.1974.tb03515.x.pd

Estimating and comparing food availability for tree-seed predators in typical pulsed-resource systems: alpine conifer forests.

Alpine conifers produce pulsed resources for the community of tree-seed consumers. Here, we describe field and statistical methods to estimate spatio-temporal variation in seed-crops in forests with different species composition. Annual and between-site variation in seed production was high and characterised by occurrence of mast-crops followed by seed-crop failure the following year. Seed-crops of different species did not fluctuate in parallel. Please see also: Plant Biosystem 143(3), Page 643 for Corrigendu

5-Thia-5-Deazaflavin, a 1e − -Transferring Flavin Analog

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65939/1/j.1432-1033.1979.tb12952.x.pd

Role of tyrosine 129 in the active site of spinach glycolate oxidase

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66136/1/j.1432-1033.1993.tb17852.x.pd

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate

Chemical Magnetoreception: Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical-Pairs

Cryptochromes (Cry) have been suggested to form the basis of light-dependent magnetic compass orientation in birds. However, to function as magnetic compass sensors, the cryptochromes of migratory birds must possess a number of key biophysical characteristics. Most importantly, absorption of blue light must produce radical pairs with lifetimes longer than about a microsecond. Cryptochrome 1a (gwCry1a) and the photolyase-homology-region of Cry1 (gwCry1-PHR) from the migratory garden warbler were recombinantly expressed and purified from a baculovirus/Sf9 cell expression system. Transient absorption measurements show that these flavoproteins are indeed excited by light in the blue spectral range leading to the formation of radicals with millisecond lifetimes. These biophysical characteristics suggest that gwCry1a is ideally suited as a primary light-mediated, radical-pair-based magnetic compass receptor