The emperor's new shibboleth.
If an idea or result can't be conveyed in plain language, how important can it really be?
Humanities↗
Biomedical subjects
Gregory A Petsko
Publications and source records attributed to Gregory A Petsko.
Twilight of a hero.
The hope for those afflicted with Parkinson's is embryonic stem cell therapy, which depends on research that the Bush administration is doing its best to strangle.
Cell Line↗
Fame is a bubble, but not for some.
Brilliant, acerbic, not given to suffer much of anybody gladly, let alone fools, Francis Crick had enormous influence that was due to his style and high scientific standards.
DNA↗
Crystal structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida, a fumarase class II type cycloisomerase: enzyme evolution in parallel pathways.
3-Carboxy-cis,cis-muconate lactonizing enzymes (CMLEs), the key enzymes in the protocatechuate branch of the beta-ketoadipate pathway in microorganisms, catalyze the conversion of 3-carboxy-cis,cis-muconate to muconolactones. We have determined the crystal structure of the prokaryotic Pseudomonas putida CMLE (PpCMLE) at 2.6 A resolution. PpCMLE is a homotetramer and belongs to the fumarase class II superfamily. The active site of PpCMLE is formed largely by three regions, which are moderately conserved in the fumarase class II superfamily, from three respective monomers. It has been proposed that residue His141, which is highly conserved in all fumarase class II enzymes and forms a charge relay with residue Glu275 (both His141 and Glu275 are in adenylosuccinate lyase numbering), acts as the general base in most fumarase class II superfamily members. However, this charge relay pair is broken in PpCMLE. The residues corresponding to His141 and Glu275 are Trp153 and Ala289, respectively, in PpCMLE. The structures of prokaryotic MLEs and that of CMLE from the eukaryotic Neurospora crassa are completely different from that of PpCMLE, indicating MLEs and CMLEs, as well as the prokaryotic and eukaryotic CMLEs, evolved from distinct ancestors, although they catalyze similar reactions. The structural differences may be related to recognition by substrates and to differences in the mechanistic pathways by which these enzymes catalyze their respective reactions.
Amino Acid Sequence↗
Spectroscopic and X-ray crystallographic characterization of bestatin bound to the aminopeptidase from Aeromonas (Vibrio) proteolytica.
Binding of the competitive, slow-binding inhibitor bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from Aeromonas proteolytica (AAP) was examined by both spectroscopic and crystallographic methods. Electronic absorption spectra of the catalytically competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the presence of bestatin revealed that both of the divalent metal ions in AAP are involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum of the [CoCo(AAP)]-bestatin complex exhibited no observable perpendicular- or parallel-mode signal. These data indicate that the two Co(II) ions in AAP are antiferromagnetically coupled yielding an S = 0 ground state and suggest that a single oxygen atom bridges between the two divalent metal ions. The EPR data obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]-bestatin complex was solved to 2.0 A resolution. Both side chains of bestatin occupy a well-defined hydrophobic pocket that is adjacent to the dinuclear Zn(II) active site. The amino acid residues ligated to the dizinc(II) cluster in AAP are identical to those in the native structure with only minor perturbations in bond length. The alkoxide oxygen of bestatin bridges between the two Zn(II) ions in the active site, displacing the bridging water molecule observed in the native [ZnZn(AAP)] structure. The M-M distances observed in the AAP-bestatin complex and native AAP are identical (3.5 A) with alkoxide oxygen atom distances of 2.1 and 1.9 A from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 A. In addition, the NH(2) group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 A. A combination of the spectroscopic and X-ray crystallographic data presented herein with the previously reported mechanistic data for AAP has provided additional insight into the substrate-binding step of peptide hydrolysis as well as insight into important small molecule features for inhibitor design.
Aeromonas↗
A drop in the bucket.
With 151 billion dollars, all world-wide AIDS programs could be fully funded for the next 12 years.
Acquired Immunodeficiency Syndrome↗
The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization.
Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.
1-Methyl-4-phenylpyridinium↗
Xylose isomerase in substrate and inhibitor michaelis states: atomic resolution studies of a metal-mediated hydride shift.
Xylose isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.95 A resolution structure of the xylitol-inhibited enzyme presented here suggests three alternative positions for the second metal ion, only one of which appears positioned in a catalytically competent manner. To complete the reaction, an active site hydroxyl species appears appropriately positioned for hydrogen transfer, as evidenced by precise bonding distances. Conversely, the 0.98 A resolution structure of the enzyme with glucose bound in the alpha-pyranose state only shows one of the metal ion conformations at the second metal ion binding site, suggesting that the linear form of the sugar is required to promote the second and third metal ion conformations. The two structures suggest a strong degree of conformational flexibility at the active site, which seems required for catalysis and may explain the poor rate of turnover for this enzyme. Further, the pyranose structure implies that His53 may act as the initial acid responsible for ring opening of the sugar to the aldose form, an observation that has been difficult to establish in previous studies. The glucose ring also appears to display significant segmented disorder in a manner suggestive of ring opening, perhaps lending insight into means of enzyme destabilization of the ground state to promote catalysis. On the basis of these results, we propose a modified version of the bridged bimetallic mechanism for hydride transfer in the case of Streptomyces olivochromogenes xylose isomerase.
Aldose-Ketose Isomerases↗
The ascent of man?
Anyone who cares about the moral and social implications of genomics, genetic engineering and biotechnology should read Michael J. Sandel's article, 'The Case Against Perfection', in the April 2004 issue of The Atlantic Monthly.
Biotechnology↗
Doctor Dunsel.
Repressingly, a robot scientist can generate functional genomic hypothesis and carry out experiments, increasing the working scientist's feelings of redundancy.
Artificial Intelligence↗
Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis.
CDP-D-glucose 4,6-dehydratase catalyzes the conversion of CDP-D-glucose to CDP-4-keto-6-deoxyglucose in an NAD(+)-dependent manner. The product of this conversion is a building block for a variety of primary antigenic determinants in bacteria, possibly implicated directly in reactive arthritis. Here, we describe the solution of the high-resolution crystal structure of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis in the resting state. This structure represents the first CDP nucleotide utilizing dehydratase of the short-chain dehydrogenase/reductase (SDR) family to be determined, as well as the first tetrameric structure of the subfamily of SDR enzymes in which NAD(+) undergoes a full reaction cycle. On the basis of a comparison of this structure with structures of homologous enzymes, a chemical mechanism is proposed in which Tyr157 acts as the catalytic base, initiating hydride transfer by abstraction of the proton from the sugar 4'-hydroxyl. Concomitant with the removal of the proton from the 4'-hydroxyl oxygen, the sugar 4'-hydride is transferred to the B face of the NAD(+) cofactor, forming the reduced cofactor and a CDP-4-keto-d-glucose intermediate. A conserved Lys161 most likely acts to position the NAD(+) cofactor so that hydride transfer is favorable and/or to reduce the pK(a) of Tyr157. Following substrate oxidation, we propose that Lys134, acting as a base, would abstract the 5'-hydrogen of CDP-4-keto-D-glucose, priming the intermediate for the spontaneous loss of water. Finally, the resulting Delta(5,6)-glucoseen intermediate would be reduced suprafacially by the cofactor, and reprotonation at C-5' is likely mediated by Lys134.
Amino Acid Sequence↗
Structural basis for differences in substrate selectivity in Kex2 and furin protein convertases.
Kex2 is the yeast prototype of a large family of serine proteases that are highly specific for cleavage of their peptide substrates C-terminal to paired basic sites. This paper reports the 2.2 A resolution crystal structure of ssKex2 in complex with an Ac-Arg-Glu-Lys-Arg peptidyl boronic acid inhibitor (R = 19.7, R(free) = 23.4). By comparison of this structure with the structure of the mammalian homologue furin [Henrich, S., et al. (2003) Nat. Struct. Biol. 10, 520-526], we suggest a structural basis for the differences in substrate recognition at the P(2) and P(4) positions between Kex2 and furin and provide a structural rationale for the lack of P(6) recognition in Kex2. In addition, several monovalent cation binding sites are identified, and a mechanism of activation of Kex2 by potassium ion is proposed.
Binding Sites↗
Good chemistry.
The subject matter in chemistry courses reflects almost nothing of the issues that chemists are interested in. It is important to formulate a set of topics--and a Medical College Admissions Test reflecting them--that would leave chemistry departments no choice but to change their teaching.
Chemistry↗
Bad chemistry.
General chemistry courses haven't changed significantly in forty years. Because most basic chemistry students are premedical students, medical schools have enormous influence and could help us start all over again to create undergraduate chemistry education that works.
Chemistry↗
The 1.8-A resolution crystal structure of YDR533Cp from Saccharomyces cerevisiae: a member of the DJ-1/ThiJ/PfpI superfamily.
The yeast gene YDR533C encodes a protein belonging to the DJ-1/ThiJ/PfpI superfamily. This family includes the human protein DJ-1, which is mutated in autosomal recessive early-onset Parkinson's disease. The function of DJ-1 and its yeast homologue YDR533Cp is unknown. We report here the crystal structure of YDR533Cp at 1.8-A resolution. The structure indicates that the closest relative to YDR533Cp is the Escherichia coli heat shock protein Hsp31 (YedU), which has both chaperone and protease activity. As expected, the overall fold of the core domain of YDR533Cp is also similar to that of DJ-1 and the bacterial protease PfpI. YDR533Cp contains a possible catalytic triad analogous to that of Hsp31 and an additional domain that is present in Hsp31 but is not seen in DJ-1 and other members of the family. The cysteine in this triad (Cys-138) is oxidized in this crystal structure, similar to modifications seen in the corresponding cysteine in the crystal structure of DJ-1. YDR533Cp appears to be a dimer both in solution and the crystal, but this dimer is formed by a different interface than that found in Hsp31 or other members of the superfamily.
Models, Molecular↗
"Sleeping beauty": quiescence in Saccharomyces cerevisiae.
The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.
Cell Survival↗
Live and let diet.
It used to be that one's generation could be defined simply by the war one fought in. Since the middle of the last century, equally defining are the diets that each generation has been swept up in.
Diet, Reducing↗