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At least 757 records · Page 42Linked to original sources
PubMed1978

Quantitative subunit hybridization of drosophila alpha-glycerophosphate dehydrogenase.

The dimeric enzyme, alpha-Glycerophosphate dehydrogenase, was purified from eight Drosophila species by the method of Collier et al. (1976). The enzymes were inactivated at high pH and the conditions sufficient for reactivation were established. Electrophoretic patterns of reactivated alpha-glycerophosphate dehydrogenases which were mixed following inactivation of two species' enzymes, demonstrate that high pH dissociates the enzyme into its constituent subunits and reactivation involves subunit reassociation. Twenty interspecific combinations of dissociated enzymes were allowed to reassociate, and the amounts of both heterospecific and homospecific enzyme activity and protein were determined by densitometry. In all 20 tests there were no differences between observed and expected heterospecific:homospecific enzyme ratios. These results are consistent with the very slow rate of evolution of this enzyme in the family Drosophilidae (Collier and MacIntyre, 1977).

Animals↗
PubMed2003

Structure and evolution of the luciferin-regenerating enzyme (LRE) gene from the firefly Photinus pyralis.

To study the structural features of genes for the luciferin-regenerating enzyme (LRE), the entire gene along with 524 bp of upstream sequence was determined from Photinus pyralis (Coleoptera: Lampyridae). The LRE gene revealed an open reading frame composed of five exons divided by four introns ranging in size from 47 to 904 bp. The deduced LRE amino acid sequence showed identity to senescence marker protein-30 (SMP30) from a number of insects and mammals including four putative SMP30 sequences from Anopheles gambiae. Gene structure comparisons showed some intron/exon site conservation with A. gambiae and mammalian SMP30 proteins but not Drosophila. LRE and luciferase sequence comparisons revealed two conserved putative luciferin-binding sites. The evolution of LRE was discussed in relation to its function.

Amino Acid Sequence↗
PubMed2000

Effect of redox mediators on nitrogenase and hydrogenase activities in Azotobacter vinelandii.

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MBo) when the concentration of this mediator in the medium is increased up to 72 microM. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NRo) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H2 uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H2 evolved/mg cell protein/min from the nitrogenase and 160 nM H2-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H2 evolution and for H2 uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.

Azotobacter vinelandii↗
PubMed1997

Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the plant biosynthetic pathway that leads to glycinebetaine. Rice plants (Oryza sativa L.), albeit considered a typical non-glycinebetaine accumulating species, have been found to express this enzyme at low levels. This observation evokes an interest in phylogenic evolution of the enzyme in the plant kingdom. It is reported here that rice plants possess the ability to take up exogenously added betaine aldehyde through the roots and convert it to glycinebetaine, resulting in an enhanced salt-tolerance of the plants. A gene encoding a putative BADH from the rice genome was also cloned and sequenced. The gene was found to contain 14 introns, and the overall nucleotide sequence of the coding region is c. 78% identical to that of the barley BADH cDNA. Cloning of a partial BADH cDNA from rice was accomplished by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence of the cloned fragment was found to be identical to the corresponding exon regions of the rice genomic BADH gene. The deduced amino acid sequences of rice and barley BADH both contain a C-terminal tripeptide SKL, a signal known to target preproteins to microbodies. This localization was confirmed by an immuno-gold labeling study of transgenic tobacco harboring barley cDNA, which showed BADH protein inside peroxisomes. Northern blot analysis revealed that the level of BADH mRNA is salt-inducible.

Aldehyde Oxidoreductases↗
PubMed2006

Divergence of duplicated genes in maize: evolution of contrasting targeting information for enzymes in the porphyrin pathway.

The divergence of sequence and expression pattern of duplicated genes provides a means for genetic innovation to occur without sacrificing an essential function. The cpx1 and cpx2 genes of maize are a singular example of duplicated genes that have diverged by deletion and creation of protein targeting information. The cpx genes encode coproporphyrinogen III oxidase ('coprogen oxidase'), which catalyzes a step in the synthesis of chlorophyll and heme. In plants, this enzyme has been found exclusively in the plastids. The cpx1 and cpx2 genes encode almost identical, catalytically active enzymes with distinctive N-terminal peptide sequences. The cpx1 gene encodes the expected plastid transit peptide, but this region is deleted from the cpx2 gene. While the 5' regions of both messenger RNAs are highly similar, the cpx2 gene has an open-reading frame that could encode a new targeting signal. GFP fused with CPX1 localized to the plastids. In contrast, the GFP fusion with CPX2 did not target plastids and appeared to localize to mitochondria. Both cpx genes are expressed ubiquitously but, based on mutant phenotype, they seem to have discrete biological roles. Seedlings homozygous for a null mutation in the cpx1 gene completely lack chlorophyll and develop necrotic lesions in the light. However, the mutant seedlings and callus cultures will grow in tissue culture in the dark, implying that they retain a capacity to produce heme. We discuss models for the evolution of the cpx genes and possible roles of mitochondrion-localized coprogen oxidase activity in maize.

Amino Acid Sequence↗
PubMed1992

Exons encoding the highly conserved part of human glutaminyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are important components of the genetic apparatus. In spite of common catalytic properties, synthetases with different amino acid specificities are widely diverse in their primary structures, subunit sizes, and subunit composition. However, synthetases with given amino acid specificities are well conserved throughout evolution. We have been studying the human glutaminyl-tRNA synthetase possessing a sequence of about 400 amino acid residues (the core region) that is very similar to sequences in the corresponding enzymes from bacteria and yeast. The conserved sequence appears to be essential for the basic function of the enzyme, the charging of tRNA with glutamine. As a first step to a better understanding of the evolution of this enzyme, we determined the coding region for the conserved part of the human glutaminyl-tRNA synthetase. The coding region is composed of eight exons. It appears that individual exons encode defined secondary structural elements as parts of functionally important domains of the enzyme. Evolution of the gene by assembly of individual exons seems to be a viable hypothesis; alternative pathways are discussed.

Amino Acid Sequence↗
PubMed2001

Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions.

We have studied the stereospecificities of various pyridoxal 5'-phosphate dependent enzymes for the hydrogen transfer between the C-4' of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Prior to our studies, pyridoxal enzymes so far studied were reported to catalyze the hydrogen transfer only on the si-face of the planar imine intermediate formed from substrate and coenzyme. This finding had been considered as the evidence that pyridoxal enzymes have evolved divergently from a common ancestral protein, because identity in the stereospecificity reflects the similarity in the active-site structure, in particular in the geometrical relationship between the coenzyme and the active site base participating in the hydrogen transfer. However, we found that D-amino acid aminotransferase, branched-chain L-amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase catalyze the re-face specific hydrogen transfer, and that amino acid racemases catalyze the nonstereospecific hydrogen transfer. These findings suggest the convergent evolution of pyridoxal enzymes. Crystallographical studies have shown that the stereospecificity reflects the active-site structure of the enzymes, and that the enzymes with the same fold exhibit the same stereospecificity. The active site structure with the catalytic base being situated on the specific face of the cofactor has been conserved during the evolution among the pyridoxal enzymes of the same family.

Binding Sites↗
PubMed1999

Catalytic promiscuity and the evolution of new enzymatic activities.

Several contemporary enzymes catalyze alternative reactions distinct from their normal biological reactions. In some cases the alternative reaction is similar to a reaction that is efficiently catalyzed by an evolutionary related enzyme. Alternative activities could have played an important role in the diversification of enzymes by providing a duplicated gene a head start towards being captured by adaptive evolution.

Bacteria↗
PubMed2002

Structural genomics of proteins from conserved biochemical pathways and processes.

During the past year, X-ray crystallographers and solution NMR spectroscopists have made significant progress towards the complete structural characterization of conserved biochemical pathways and processes. Some of these advances were made in the context of nascent structural genomics programs, which promise to accelerate structural studies of biologically and medically important proteins. The results of high-throughput protein production, crystallization, structure determination, homology modeling and functional annotation published by two such programs have provided insight into the evolution and function of enzymes in the isoprenoid biosynthesis and ribulose monophosphate pathways.

Biological Evolution↗
PubMed2001

How does a DNA interacting enzyme change its specificity during molecular evolution? A site-directed mutagenesis study at the DNA binding site of the DNA-(adenine-N6)-methyltransferase EcoRV.

The EcoRV DNA-(adenine-N6)-methyltransferase (MTase) recognizes GATATC sequences and modifies the first adenine residue within this site. Parts of its DNA interface show high sequence homology to DNA MTases of the dam family which recognize and modify GATC sequences. A phylogenetic analysis of M.EcoRV and dam-MTases suggests that EcoRV arose in evolution from a primordial dam-MTase in agreement to the finding that M.EcoRV also methylates GATC sites albeit at a strongly reduced rate. GATCTC sites that deviate in only one position from the EcoRV sequence are preferred over general dam sites. We have investigated by site-directed mutagenesis the function of 17 conserved and nonconserved residues within three loops flanking the DNA binding cleft of M.EcoRV. M.EcoRV contacts the GATATC sequence with two highly cooperative recognition modules. The contacts to the GAT-part of the recognition sequence are formed by residues conserved between dam MTases and M.EcoRV. Mutations at these positions lead to an increase in the discrimination between GATATC and GATC substrates. Our data show that the change in sequence specificity from dam (GATC) to EcoRV (GATATC) was accompanied by the generation of a second recognition module that contacts the second half of the target sequence. The new DNA contacts are formed by residues from all three loops that are not conserved between M.EcoRV and dam MTases. Mutagenesis at important residues within this module leads to variants that show a decreased ability to recognize the TC-part of the GATATC sequence.

Amino Acid Sequence↗