Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutants proteins--His176----Asn, Cys149----Ser, Cys149----Gly--were isolated from one or both of the enzymes. The study of the properties of these mutants has shown that Cys149 is clearly responsible for the information of a charge-transfer transition, named the Racker band, observed during the NAD+ binding to apoGAPDH. This result excludes a similarity between the Racker band and the charge-transfer transition observed following the alkylation of GAPDH by 3-chloroacetyl pyridine-adenine dinucleotide.     

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.     

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl-beta-gamma-imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Glyceraldehyde-3-phosphate dehydrogenase

Conflicting experimental evidence of the pathway of catalysis for the enzyme from rabbit, pig and lobster muscle tissues is reviewed. Transient kinetic studies with the enzyme from rabbit muscle are presented. The results are shown to be consistent with the double-displacement mechanism of catalysis originally proposed by Segal & Boyer (1953). The rate constant for combination of the aldehyde form of the substrate with the NAD+ complex of the enzyme is about 3 X 10(7) M-1 S-1, and for all four subunits of the molecule the rate constant for hydride transfer in the ternary complex formed is greater than 10(3) S-1, consistent with their simultaneous participation in catalysis. Recent steady-state kinetic studies with the rabbit muscle enzyme, in contrast to earlier studies, also provide evidence to support the Segal-Boyer pathway if the kinetic effects of the negative cooperativity of NAD+ binding are taken into account. Experimental data for the binding of NAD+ to the enzyme from muscles and from Bacillus stearothermophilus, and their interpretations, are also briefly reviewed. The information currently available from X-ray crystallography regarding the structures of holoenzyme and apoenzyme from B. stearothermophilus and lobster muscle is outlined.     

X-ray crystal structure of human dopamine sulfotransferase, SULT1A3. Molecular modeling and quantitative structure-activity relationship analysis demonstrate a molecular basis for sulfotransferase substrate specificity

Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.