Using conventional and scanning transmission electron microscopy, we have examined the physical basis of long-range enhancer effects between distal and proximal elements in a eukaryotic promoter. Specifically, we have studied binding of human transcription factor Sp1 to 10-base-pair G+C-rich elements ("GC boxes") located at -100 and +1700 relative to the RNA start site. It was previously observed that the distantly located site functions in synergism with the promoter-proximal site to strongly activate transcription in vivo. Here we demonstrate that this synergism is likely to be a direct consequence of interactions between remote and local Sp1, the remote Sp1 translocated to the promoter by a DNA loop. Scanning transmission electron microscopy shows that Sp1 initially forms a tetramer and subsequently assembles multiple tetramers stacked in register at the DNA loop juncture. This unexpected finding not only provides the physical basis for loop formation but also defines a biological process leading to strongly increased concentration of activator protein at the promoter. The mechanism may unify the problem of transcriptional activation by removing enhancer action as a separate class of regulatory activity.

https://www.pnas.org/content/pnas/88/13/5670.full.pdf

DNA looping and Sp1 multimer links: a mechanism for transcriptional synergism and enhancement.

The most recent research on D-amino acid oxidases and D-amino acid metabolism has revealed new, intriguing properties of flavoenzymes and enlighted novel biotechnological uses of this catalyst Concerning the in vivo function of the enzyme, new findings on the physiological role of D-amino acid oxidase point to a detoxifying function of the enzyme in metabolizing exogenous D-amino acids in animals A novel role in modulating the level of D-serine in brain has also been proposed for the enzyme At the molecular level, site-directed mutagenesis studies on the pig kidney D-amino acid oxidase and, more recently, on the enzyme from the yeast Rhodotorula gracilis indicated that the few conserved residues of the active site do not play a role in acid-base catalysis but rather are involved in substrate interactions The three-dimensional structure of the enzyme was recently determined from two different sources: at 25–30 A resolution for DAAO from pig kidney and at 12–18 A resolution for R gracilis The active site can be clearly depicted: the striking absence of essential residues acting in acid-base catalysis and the mode of substrate orientation into the active site, taken together with the results of free-energy correlation studies, clearly support a hydrid transfer type of mechanism in which the orbital steering between the substrate and the isoalloxazine atoms plays a crucial role during catalysis

D-Amino acid oxidase: new findings.

Cellobiose dehydrogenase catalyses the oxidation of aldoses--a simple reaction, a boring enzyme? No, neither for the envisaged bioelectrochemical applications nor mechanistically. The catalytic cycle of this flavocytochrome is complex and modulated by its flexible cytochrome domain, which acts as a built-in redox mediator. This intramolecular electron transfer is modulated by the pH, an adaptation to the environmental conditions encountered or created by the enzyme-producing fungi. The cytochrome domain forms the base from which electrons can jump to large terminal electron acceptors, such as redox proteins, and also enables by that path direct electron transfer from the catalytically active flavodehydrogenase domain to electrode surfaces. The application of electrochemical techniques to the elucidation of the molecular and catalytic properties of cellobiose dehydrogenase is discussed and compared to biochemical methods. The results lead to valuable insights into the function of this cellulose-bound enzyme, but also form the basis of exciting applications in biosensors, biofuel cells and bioelectrocatalysis.

Cellobiose Dehydrogenase: A Versatile Catalyst for Electrochemical Applications.

The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4(2-) derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refined phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound beta-barrel of the parallel alpha/beta class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other beta-barrel structures, the known active-site residues are located in the center of the beta-barrel and are accessible to substrate from the COOH side of the beta-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the beta-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.

https://www.pnas.org/content/pnas/84/22/7846.full.pdf

Molecular architecture of rabbit skeletal muscle aldolase at 2.7-A resolution.

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S C, Gerlt, J A, Powers, V M, & Kenyon, G L (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA) Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P putida (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli.

The structure of flavocytochrome b2 from baker's yeast was solved at 3.0-A resolution by the multiple isomorphous replacement method combined with solvent leveling procedures, using data collected from an area detector. The tetramer of Mr 230,000 has 4-fold symmetry. Each subunit contains a cytochrome domain consisting of the first 100 residues, a flavin-binding domain containing the next 386 residues, and an extended C-terminal tail of 25 residues. The cytochrome domain closely resembles microsomal cytochrome b5, whereas the flavin-binding domain contains a parallel beta 8/alpha 8 barrel motif similar to glycolate oxidase and trimethylamine dehydrogenase. Two of the four cytochrome domains are disordered in the crystals. The flavin ring and heme group are separated by about 16 A between their centers, and their planes are inclined by about 17 degrees to each other.

Three-dimensional structure of flavocytochrome b2 from baker's yeast at 3.0-A resolution

The structure of p-cresol methylhydroxylase [4-cresol:(acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1], a flavocytochrome c, has been determined at 6.0-A resolution. The structure analysis is based on two heavy-atom derivatives with anomalous scattering and 2-fold averaging about a noncrystallographic axis. The molecule is an alpha 2 beta 2 tetramer with a cytochrome subunit of Mr approximately 8500 and a flavoprotein subunit of Mr approximately 49,000. The flavoprotein subunits are tightly packed about the molecular 2-fold axis, whereas the cytochrome subunits are located on the outside of the molecule, each in a depression on the surface of a flavoprotein subunit. The results of this study have led to the following conclusions. The alpha 2 beta 2 quaternary structure of the enzyme is different from alpha beta as originally thought. The orientation of the cytochrome subunit and the surface complementarity of the cytochrome and flavoprotein subunits are clearly defined. The cytochrome subunit is similar in size to other small bacterial cytochromes but probably forms a distinct subclass. The titration (by substrate) behavior of the enzyme and other kinetic properties are rationalized by its quaternary structure.

Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution.

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Three-dimensional structure of flavocytochrome b2 from baker's yeast at 3.0-A resolution

Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution.