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M M Stayton

Publications and source records attributed to M M Stayton.

12 recordsLinked to original sources
PubMed1999

Adenylosuccinate synthetase: recent developments.

By exerting strategic control on purine nucleotide biosynthesis, and by engaging GTP-dependent transphosphorylation of IMP to activate loss of an oxygen atom during catalysis, adenylosuccinate synthetase remains as enzyme that justifiably fascinates students of enzyme catalysis. This review describes how the balanced application of X-ray crystallography and enzyme kinetics has advanced the comprehension of the catalytic and regulatory properties of adenylosuccinate synthetase. Detailed analysis has demonstrated the formation of 6-phosphoryl-IMP, an intermediate originally postulated over 40 years ago on the basis of oxygen-18 exchange experiments showing that position-6 oxygen of IMP becomes incorporated into phosphate. Inferences about the participation of amino acid side-chains that stabilize 6-P-IMP during catalysis have also been confirmed by site-directed mutagenesis and examination of such mutations on various kinetic parameters. Moreover, the action of certain regulatory ligands have also been viewed at atomic level resolution. For example, magnesium ion and GDP can induce conformational changes linked to the stabilization of one of two known conformations of the so-called 40s loop. Another significant finding is that two magnesium ions play fundamental roles: one binding with high affinity to the substrate GTP, and a second binding with lower affinity to the co-substrate aspartate. These structural and kinetic studies have also formed the basis for clarifying the action of various inhibitors and potentially important pharmacologic agents with this key regulatory enzyme. Finally, this review explores the current status of investigations on gene structure and gene expression in a number of organisms.

Adenylosuccinate Synthase↗
PubMed1997

Avirulence gene D of Pseudomonas syringae pv. tomato may have undergone horizontal gene transfer.

Avirulence gene D (avrD) is carried on the B-plasmid of the plant pathogen Pseudomonas syringae pv. tomato with plasmid-borne avrD homologs widely distributed among the Pseudomonads. We now report sequences in the soft rot pathogen Erwinia carotovora that cross-hybridize to avrD suggesting a conserved function beyond avirulence. Alternatively, avrD may have been transferred horizontally among species: (i) DNA linked to avrD shows evidence of class II transpositions and contains a novel IS3-related insertion sequence, and (ii) short sequences linked to avrD are similar to pathogenicity genes from a variety of unrelated pathogens. We have also identified the gene cluster that controls B-plasmid stability.

Bacterial Proteins↗
PubMed1993

High-level expression of porcine fructose-1,6-bisphosphatase in Escherichia coli: purification and characterization of the enzyme.

A cDNA encoding porcine fructose-1,6-bisphosphatase was isolated from total pig liver RNA. The enzyme's coding sequence was fused to the bacteriophage T7 gene 10 promoter and transcription terminator sequence and expressed in E. coli under control of the T7 RNA polymerase. Induced cells contain elevated levels of fructose-1,6-bisphosphatase enzymatic activity and an abundant 37,000 dalton protein. The enzyme was purified to apparent homogeneity and judged identical to wild-type porcine fructose-1,6-bisphosphatase. The kinetic parameters are similar to those reported for the pig kidney enzyme. The N-terminal amino acid sequence is identical to the predicted sequence and the kinetic parameters are consistent with freedom from proteolysis. As estimated from enzymatic activity and visual inspection of coomassie blue-stained SDS-PAGE gels, porcine fructose-1,6-bisphosphatase constitutes as much as 20% of the soluble protein from the over-expressing E. coli strain.

Amino Acid Sequence↗
PubMed1989

Photosynthetic Genes of Petunia (Mitchell) Are Differentially Expressed during the Diurnal Cycle.

The petunia (Petunia [Mitchell]) chloroplast proteins, the chlorophyll a/b-binding (Cab) proteins, and the small subunit of ribulose bisphosphate carboxylase (RbcS) are encoded by nuclear genes that are expressed in a light-dependent manner. The steady-state concentrations of five cab mRNAs vary with a dramatic circadian rhythm in plants grown under a constant diurnal cycle (10 hours light, 14 hours dark). cab mRNA levels reach their maximum during the light period, but begin to drop prior to the dark period. These RNAs fall to their minimum concentration during the dark period and then begin to increase again in anticipation of the light. Within this general pattern, there are variations in expression among specific classes of cab genes. The light harvesting complex of photosystem II LHCII-type 1 cab mRNAs rise to a well-defined maximum at 2 hours prior to the dark period. All but one of these genes are expressed in anticipation of the light period. The LHCII type 2 cab mRNA and the LHC of photosystem I cab mRNA are expressed at more constant levels throughout the light period. The expression of these genes anticipates the light more than does the expression of the LHCII type 1 genes. The steady state mRNA levels for the petunia rbcS genes show no significant diurnal fluctuation.

Journal Article↗
PubMed1987

Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli.

Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines. A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45. Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level. A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism. The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria. The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme. Michaelis constants for both the wild-type and overproduced enzyme preparations were the same. And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.

Adenylosuccinate Synthase↗
PubMed1986

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

Amino Acid Sequence↗
PubMed1983

Complexes of Escherichia coli primase with the replication origin of G4 phage DNA.

Escherichia coli primase (dnaG protein), an essential DNA replication enzyme, synthesizes a primer at the unique origin sequence of the single-stranded circular phage G4 DNA (Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 758-764). Kinetic analyses suggest that for each DNA molecule at least two primase molecules participate in the reaction. Binding of 3H-labeled primase is specific for the G4 complementary strand origin region and is saturated at approximately 2 primase molecules/DNA circle. Such complexes, isolated by gel filtration, function in the absence of additional primase to convert the phage DNA to the duplex form. Although the primase-DNA complex is stable to refiltration, the DNA-bound enzyme can dissociate and reattach to function at the origin sequence of another G4 DNA circle. An antibody to primase blocks the action of primase in the free form or within a DNA complex and even interferes with extension of the primer by DNA polymerase III holoenzyme. These kinetic and binding studies of G4 priming, the least complicated of the primase systems, suggest that 2 primase molecules form a complex at the origin region and remain bound even after transcribing a sequence to prime DNA replication.

Coliphages↗
PubMed1979

Purification, properties, and kinetics of D-ribulokinase from Aerobacter aerogenes.

The enzyme D-ribulokinase from Aerobacter aerogenes was purified to near homogeneity. The molecular weight, as determined by Sephacryl gel chromatography, is 116,000. The subunit molecular weight, determined by sodium dodecyl sulfate-gel electrophoresis, is 59,000, suggesting that D-ribulokinase is a dimer of identical subunits. Initial rate kinetic studies, involving substrate analogs and products, were carried out. These investigations support a kinetic mechanism of the Random Bi Bi type. Isotope partitioning, utilizing D-[3H]ribulose, indicates that the mechanism is steady state Random Bi Bi.

Enterobacter↗
PubMed1979

Guanosine 5'-diphosphate-3'-diphosphate inhibition of adenylosuccinate synthetase.

The mechanism of ppGpp inhibition of adenylosuccinate synthetase (EC 6.3.4.4) was examined. Initial rate kinetic studies demonstrate the ppGpp inhibition is competitive with respect to GTP and noncompetitive with respect to L-aspartate and IMP. This is in contrast to an earlier report (Gallant, J., Irr, J., and Cashel, M. (1971) J. Biol. Chem. 246, 5812-5816), which suggested that ppGpp did not bind at the GTP site. Possible reasons for the discrepancy are discussed. The potency of the ppGpp inhibition is confirmed.

Adenylosuccinate Synthase↗