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Gregory A Petsko

Publications and source records attributed to Gregory A Petsko.

88 records · Page 5Linked to original sources
PubMed2002

Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.

Adenosine Diphosphate↗
PubMed2002

The guards themselves.

If we agree that, in the present climate of fear of bioterrorism, some restrictions on the conduct and/or publication of certain types of biological research are likely, it is to our advantage to preempt government action by devising for ourselves restrictions that we can live with, then the inevitable question becomes: how should these restrictions be administered?

Bioterrorism↗
PubMed2002

An Asilomar moment.

As governments in the US and Europe contemplate legislation that would divert funding of some genomics-driven research to Offices of Homeland Security and the like, and that would restrict the freedom of biologists to publish and share some of their data, we of the scientific community are facing a crisis, brought on by fears of bioterrorism, that eerily mirrors the early days of recombinant DNA research.

Access to Information↗
PubMed2002

Misfolded proteins are competent to mediate a subset of the responses to heat shock in Saccharomyces cerevisiae.

Cells may sense heat shock via the accumulation of thermally misfolded proteins. To explore this possibility, we determined the effect of protein misfolding on gene expression in the absence of temperature changes. The imino acid analog azetidine-2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We found that adding AZC to yeast at sublethal concentrations sufficient to arrest proliferation selectively induced expression of heat shock factor-regulated genes to a maximum of 27-fold and that these inductions were dependent on heat shock factor. AZC treatment also selectively repressed expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20-fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins. Induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activated heat shock factor, but treatment with canavanine, an arginine analog less potent than AZC at misfolding proteins, did not. However, misfolded proteins did not strongly induce the stress response element regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock.

Azetidinecarboxylic Acid↗
PubMed2002

Fish tale.

The draft sequence of the genome of the Japanese pufferfish has just been announced and the temptation to humor is great.

Animals↗
PubMed2002

No place like Ome.

Of the two components of the word genome, 'ome' is the more interesting.

Genomics↗
PubMed2002

Our own petards.

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Biological Warfare↗
PubMed2002

Grain of truth.

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Base Pairing↗
PubMed2002

Why protein R-factors are so large: a self-consistent analysis.

The R-factor and R-free are commonly used to measure the quality of protein models obtained in X-ray crystallography. Well-refined protein structures usually have R-factors in the range of 20-25%, whereas intrinsic errors in the experimental data are usually around 5%. We use molecular dynamics simulations to perform a self-consistent analysis by which we determine the major factors contributing to large values of protein R-factors. The analysis shows that significant R-factor values can arise from the use of isotropic B-factors to model anisotropic protein motions and from coordinate errors. Even in the absence of coordinate errors, the use of isotropic B-factors can cause the R-factors to be around 10%; for coordinate errors smaller than 0.2 A, the two errors types make similar contributions. The inaccuracy of the energy function used and multistate protein dynamics are unlikely to make significant contributions to the large R-factors.

Animals↗
PubMed2002

Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion.

We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from rat liver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to a resolution of 2.3 A. In addition to the thiolester substrate, the catalytic water, which is added in the hydration reaction, has been modeled into well-defined electron density in each of the six active sites of the physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu(144) and Glu(164) of the enzyme and has a lone pair of electrons poised to react with C(3) of the enzyme-bound alpha,beta-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent of the enolase active site. However, unlike enolase, which has a lysine available to donate a proton, there are no other sources of protons available from other active site residues in ECH. Furthermore, an analysis of the hydrogen-bonding network of the active site suggests that both Glu(144) and Glu(164) are ionized and carry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule, leads to a hypothesis that the three atoms from a single water molecule are added across the double bond to form the hydrated product. The structural results are discussed in connection with details of the mechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy of enzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to the previous interpretation, the structure of the E-S complex together with previously determined kinetic isotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.

Animals↗
PubMed2002

Purification, crystallization and molecular symmetry of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis.

The enzyme CDP-D-glucose 4,6-dehydratase (EC 4.2.1.45) is an NAD(+)-dependent oxidoreductase which catalyzes the irreversible conversion of CDP-D-glucose to CDP-4-keto-6-deoxy-D-glucose. The product of this reaction is an intermediate in the synthesis of all CDP-linked 3,6-dideoxyhexoses, an important class of antigenic determinants found in the lipopolysaccharide layer of Gram-negative bacteria. Crystals of a recombinant form of this enzyme from Yersinia pseudotuberculosis have been grown in two crystal forms, both possessing pseudo-translational non-crystallographic symmetry, with dramatically different diffraction characteristics. A complete 1.8 A data set has been collected from the primitive orthorhombic crystal form, for which the non-crystallographic symmetry is described in detail.

Crystallization↗
PubMed2002

The 1.20 A resolution crystal structure of the aminopeptidase from Aeromonas proteolytica complexed with tris: a tale of buffer inhibition.

The aminopeptidase from Aeromonas proteolytica (AAP) is a bridged bimetallic enzyme that removes the N-terminal amino acid from a peptide chain. To fully understand the metal roles in the reaction pathway of AAP we have solved the 1.20 A resolution crystal structure of native AAP (PDB ID = 1LOK). The high-quality electron density maps showed a single Tris molecule chelated to the active site Zn(2+), alternate side chain conformations for some side chains, a sodium ion that mediates a crystal contact, a surface thiocyanate ion, and several potential hydrogen atoms. In addition, the high precision of the atomic positions has led to insight into the protonation states of some of the active site amino acid side chains.

Aeromonas↗
PubMed2002

Defining the mandate of proteomics in the post-genomics era: workshop report.

Research in proteomics is the next step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing genomes. This will require new technologies but it is highly likely that many of these will be developed. Looking back 10 to 20 years from now, the question is: Will we have done the job wisely or wastefully? This report summarizes the presentations made at a symposium at the National Academy of Sciences on February 25, 2002.

Computational Biology↗
PubMed2001

A christmas carol.

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Antineoplastic Agents↗