Biomedical subjects

D O Somers

Publications and source records attributed to D O Somers.

7 recordsLinked to original sources

PubMed1999

2.0 A X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue.

The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents.

Adenosine Triphosphate↗

PubMed1998

Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration.

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Crystallization↗

PubMed1997

Crystal structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase.

Sulfonamides were amongst the first clinically useful antibacterial agents to be discovered. The identification of sulfanilamide as the active component of the dye Prontosil rubrum led to the synthesis of clinically useful analogues. Today sulfamethoxazole (in combination with trimethoprim), is used to treat urinary tract infections caused by bacteria such as Escherichia coli and is also a first-line treatment for pneumonia caused by the fungus Pneumocystis carinii, a common condition in AIDS patients. The site of action is the de novo folate biosynthesis enzyme dihydropteroate synthase (DHPS) where sulfonamides act as analogues of one of the substrates, para-aminobenzoic acid (pABA). We report here the crystal structure of E.coli DHPS at 2.0 A resolution refined to an R-factor of 0.185. The single domain of 282 residues forms an eight-stranded alpha/beta-barrel. The 7,8-dihydropterin pyrophosphate (DHPPP) substrate binds in a deep cleft in the barrel, whilst sulfanilamide binds closer to the surface. The DHPPP ligand site is highly conserved amongst prokaryotic and eukaryotic DHPSs.

Amino Acid Sequence↗

PubMed1994

Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution.

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

Crystallography, X-Ray↗

PubMed1992

Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction.

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

Amino Acid Sequence↗

PubMed1991

The structure of 6-phosphogluconate dehydrogenase refined at 2.5 A resolution.

The three-dimensional structure of ovine 6-phosphogluconate dehydrogenase, refined at 2.5 A resolution with a residual for all data of 18.5%, is reported. This model, based on improved diffraction data and a corrected sequence, supersedes that reported earlier. Each subunit of the dimer has three domains: a beta-alpha-beta domain binds NADP; an all alpha domain provides much of the dimer interface; the C-terminal tail burrows into the second subunit.

Molecular Structure↗

PubMedDate not supplied

A two-step purification procedure for sheep liver 6-phosphogluconate dehydrogenase.

A two-step procedure for the purification of 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGDH) from sheep liver is described. The enzyme is directly bound to cellulose phosphate by batch extraction and eluted with a linear salt gradient. Purification is completed by affinity chromatography using NADP(+)-agarose. The result is 6-PGDH of high purity, greatly increased yield, and the highest specific activity yet achieved, with a significant reduction in the purification time.

Animals↗