scholarly journals A comparison of the enzymological and biophysical properties of two distinct classes of dehydroquinase enzymes

1992 ◽  
Vol 282 (3) ◽  
pp. 687-695 ◽  
Author(s):  
C Kleanthous ◽  
R Deka ◽  
K Davis ◽  
S M Kelly ◽  
A Cooper ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Weighted Average ◽  
Alpha Helix ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Scanning Calorimetry ◽  
Typical Type

This paper compares the biophysical and mechanistic properties of a typical type I dehydroquinase (DHQase), from the biosynthetic shikimate pathway of Escherichia coli, and a typical type II DHQase, from the quinate pathway of Aspergillus nidulans. C.d. shows that the two proteins have different secondary-structure compositions; the type I enzyme contains approx. 50% alpha-helix while the type II enzyme contains approx. 75% alpha-helix. The stability of the two types of DHQase was compared by denaturant-induced unfolding, as monitored by c.d., and by differential scanning calorimetry. The type II enzyme unfolds at concentrations of denaturant 4-fold greater than the type I and through a series of discrete transitions, while the type I enzyme unfolds in a single transition. These differences in conformational stability were also evident from the calorimetric experiments which show that type I DHQase unfolds as a single co-operative dimer at 57 degrees C whereas the type II enzyme unfolds above 82 degrees C and through a series of transitions suggesting higher orders of structure than that seen for the type I enzyme. Sedimentation and Mr analysis of both proteins by analytical ultracentrifugation is consistent with the unfolding data. The type I DHQase exists predominantly as a dimer with Mr = 46,000 +/- 2000 (a weighted average affected by the presence of monomer) and has a sedimentation coefficient s0(20,w) = 4.12 (+/- 0.08) S whereas the type II enzyme is a dodecamer, weight-average Mr = 190,000 +/- 10,000 and has a sedimentation coefficient, s0(20,w) = 9.96 (+/- 0.21) S. Although both enzymes have reactive histidine residues in the active site and can be inactivated by diethyl pyrocarbonate, the possibility that these structurally dissimilar enzymes catalyse the same dehydration reaction by the same catalytic mechanism is deemed unlikely by three criteria: (1) they have very different pH/log kcat. profiles and pH optima; (2) imine intermediates, which are known to play a central role in the mechanism of type I enzymes, could not be detected (by borohydride reduction) in the type II enzyme; (3) unlike Schiff's base-forming type I enzymes, there are no conserved lysine residues in type II amino acid sequences.

scholarly journals Amino acid sequences of α-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment

1978 ◽  
Vol 173 (2) ◽  
pp. 373-385 ◽  
Author(s):  
K H Gough ◽  
A S Inglis ◽  
W G Crewther
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Coiled Coil ◽  
Alpha Helix ◽  
Protein S ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Sequencing Data ◽  
Helical Segment

The amino acid sequence of a type-I helical segment from the low-sulphur protein (S-carboxymethylkerateine-A) of wool was determined by combining automatic and manual-sequencing data. Whereas in the type-II helical segment most of the cationic groups occur in pairs, 11 of the 22 anionic residues in the sequence of the type-I segment were situated next to a second anionic residue. This suggests possible interactions between type-I and type-II helical segments in alpha-keratin. As observed with the sequence of a type-II helical segment a model constructed on 3.6 residues per turn of helix shows a line of hydrophobic residues along the helix, thereby supporting the physicochemical evidence that the molecule is predominantly helical and forms part of a coiled-coil structure. Examination of the sequence data by predictive methods indicates the possibilty of extensive sections of alpha-helix interspersed with discontinuities. The molecule contains a number of regions with peptide sequences identical with those found by other workers after enzymic digestion of fractions from oxidized wool.

Transport Properties of Type II Sodium-Silicon Clathrates

2005 ◽  
Vol 886 ◽  
Author(s):  
Matt Beekman ◽  
Jan Grkyo ◽  
George S. Nolas
Keyword(s):  
Thermal Conductivity ◽  
Sodium Content ◽  
Type I ◽  
Type Ii ◽  
Conductivity Measurements ◽  
Scanning Calorimetry ◽  
Resistivity Measurements ◽  
Silicon Clathrates ◽  
Covalently Bonded ◽  
First Time

ABSTRACTWe have synthesized the type II silicon clathrates Na1Si136 and Na8Si136, and report on the electrical and thermal transport in these materials. The crystal structure consists of a covalently bonded silicon framework in which sodium guest atoms are encapsulated inside the silicon host framework. Differential scanning calorimetry measurements show the compounds decompose above 600°C to diamond-structure silicon. Temperature dependant electrical resistivity measurements show the specimens to have an insulating character, with magnitudes that decrease with increasing sodium content. For the first time, thermal conductivity measurements on type II sodium-silicon clathrates are presented. The thermal conductivity is very low for both specimens, and for Na8Si136 exhibits a clear dip in the range from 50 to 70 K. These data suggest that the “rattling” behavior observed in type I clathrates may also be present in type II clathrates.

scholarly journals The lspA Gene, Encoding the Type II Signal Peptidase of Rickettsia typhi: Transcriptional and Functional Analysis

2006 ◽  
Vol 189 (2) ◽  
pp. 336-341 ◽  
Author(s):  
M. Sayeedur Rahman ◽  
Shane M. Ceraul ◽  
Sheila M. Dreher-Lesnick ◽  
Magda S. Beier ◽  
Abdu F. Azad
Keyword(s):  
Amino Acid Sequences ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
Transcriptional Level ◽  
Temperature Sensitive ◽  
Type I ◽  
Type Ii ◽  
Intracellular Growth ◽  
Rickettsia Typhi ◽  
Differential Expression Pattern

ABSTRACT Lipoprotein processing by the type II signal peptidase (SPase II) is known to be critical for intracellular growth and virulence for many bacteria, but its role in rickettsiae is unknown. Here, we describe the analysis of lspA, encoding a putative SPase II, an essential component of lipoprotein processing in gram-negative bacteria, from Rickettsia typhi. Alignment of deduced amino acid sequences shows the presence of highly conserved residues and domains that are essential for SPase II activity in lipoprotein processing. The transcription of lspA, lgt (encoding prolipoprotein transferase), and lepB (encoding type I signal peptidase), monitored by real-time quantitative reverse transcription-PCR, reveals a differential expression pattern during various stages of rickettsial intracellular growth. The higher transcriptional level of all three genes at the preinfection time point indicates that only live and metabolically active rickettsiae are capable of infection and inducing host cell phagocytosis. lspA and lgt, which are involved in lipoprotein processing, show similar levels of expression. However, lepB, which is involved in nonlipoprotein secretion, shows a higher level of expression, suggesting that LepB is the major signal peptidase for protein secretion and supporting our in silico prediction that out of 89 secretory proteins, only 14 are lipoproteins. Overexpression of R. typhi lspA in Escherichia coli confers increased globomycin resistance, indicating its function as SPase II. In genetic complementation, recombinant lspA from R. typhi significantly restores the growth of temperature-sensitive E. coli Y815 at the nonpermissive temperature, supporting its biological activity as SPase II in prolipoprotein processing.

scholarly journals Amino acid sequences of α-helical segments from S-carboxymethylkerateine-A. Complete sequence of a type-II segment

1978 ◽  
Vol 173 (2) ◽  
pp. 365-371 ◽  
Author(s):  
W G Crewther ◽  
A S Inglis ◽  
N M McKern
Keyword(s):  
Amino Acid ◽  
Coiled Coil ◽  
Complete Sequence ◽  
Amino Acid Sequences ◽  
Helical Axis ◽  
Type I ◽  
Type Ii ◽  
Coil Structure ◽  
Aqueous Urea ◽  
Alpha Keratin

1. The helical fragments obtained by partial chymotryptic digestion of S-carboxymethylkeratine-A, the low-sulphur fraction from wool, were fractionated into type-I and type-II helical segments in aqueous urea under conditions limiting carbamoylation. 2. The amino acid sequence of a 109-residue type-II segment was completed by using the sequenator. 3. When the data were incorporated into a helical model of 3.6 residues per turn the hydrophobic residues generated a band aligned at a slight angle to the helical axis. This result is in accord with the postulated coiled-coil structure of the crystalline regions of alpha-keratin.

Sequence and expression of a type II keratin, K5, in human epidermal cells

1988 ◽  
Vol 8 (1) ◽  
pp. 486-493
Author(s):  
R Lersch ◽  
E Fuchs
Keyword(s):  
Human Gene ◽  
Basal Layer ◽  
Epidermal Cells ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Human Epidermal Cells ◽  
Rna Blot Analysis

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.

scholarly journals Sequence and expression of a type II keratin, K5, in human epidermal cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 486-493 ◽  
Author(s):  
R Lersch ◽  
E Fuchs
Keyword(s):  
Human Gene ◽  
Basal Layer ◽  
Epidermal Cells ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Gene Encoding ◽  
Human Epidermal Cells ◽  
Rna Blot Analysis

We report here the cDNA and amino acid sequences of a human 58-kilodalton type II keratin, K5, which is coexpressed with a 50-kilodalton type I keratin partner, K14, in stratified squamous epithelia. Using a probe specific for the 3'-noncoding portion of this K5 cDNA, we demonstrated the existence of a single human gene encoding this sequence. Using Northern (RNA) blot analysis and in situ hybridization with cRNA probes for both K5 and K14, we examined the expression of these mRNAs in the epidermis and in cultured epidermal cells. Our results indicate that the mRNAs for K5 and K14 are coordinately expressed and abundant in the basal layer of the epidermis. As cells undergo a commitment to terminally differentiate, the expression of both mRNAs seems to be downregulated.

NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1301-1313 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Peter Ricke ◽  
Werner Liesack
Keyword(s):  
16S Rrna ◽  
Amino Acid Sequences ◽  
Phenotypic Trait ◽  
Type I ◽  
Methanotrophic Bacteria ◽  
Type Ii ◽  
Methylococcus Capsulatus ◽  
Available Nitrogen ◽  
Active Growth ◽  
Nitrogen Acquisition

The ability to utilize dinitrogen as a nitrogen source is an important phenotypic trait in most currently known methanotrophic bacteria (MB). This trait is especially important for acidophilic MB, which inhabit acidic oligotrophic environments, highly depleted in available nitrogen compounds. Phylogenetically, acidophilic MB are most closely related to heterotrophic dinitrogen-fixing bacteria of the genus Beijerinckia. To further explore the phylogenetic linkage between these metabolically different organisms, the sequences of nifH and nifD gene fragments from acidophilic MB of the genera Methylocella and Methylocapsa, and from representatives of Beijerinckia, were determined. For reference, nifH and nifD sequences were also obtained from some type II MB of the alphaproteobacterial Methylosinus/Methylocystis group and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences of nifH and nifD were highly congruent. The phylogenetic relationships among MB in the NifH and NifD trees also agreed well with the corresponding 16S rRNA-based phylogeny, except for two distinctive features. First, different methods used for phylogenetic analysis grouped the NifH and NifD sequences of strains of the gammaproteobacterial MB Methylococcus capsulatus within a clade mainly characterized by Alphaproteobacteria, including acidophilic MB and type II MB of the Methylosinus/Methylocystis group. From this and other genomic data from Methylococcus capsulatus Bath, it is proposed that an ancient event of lateral gene transfer was responsible for this aberrant branching. Second, the identity values of NifH and NifD sequences between Methylocapsa acidiphila B2 and representatives of Beijerinckia were clearly higher (98·5 and 96·6 %, respectively) than would be expected from their 16S rRNA-based relationships. Possibly, these two bacteria originated from a common acidophilic dinitrogen-fixing ancestor, and were subject to similar evolutionary pressure with regard to nitrogen acquisition. This interpretation is corroborated by the observation that, in contrast to most other diazotrophs, M. acidiphila B2 and Beijerinckia spp. are capable of active growth on nitrogen-free media under fully aerobic conditions.

scholarly journals Characterization of the 3-dehydroquinase domain of the pentafunctional AROM protein, and the quinate dehydrogenase from Aspergillus nidulans, and the overproduction of the type II 3-dehydroquinase from neurospora crassa

1993 ◽  
Vol 296 (2) ◽  
pp. 451-457 ◽  
Author(s):  
A R Hawkins ◽  
J D Moore ◽  
A M Adeokun
Keyword(s):  
Neurospora Crassa ◽  
Aspergillus Nidulans ◽  
Shikimate Pathway ◽  
Research Programme ◽  
Quinic Acid ◽  
Type I ◽  
Type Ii ◽  
Typical Type ◽  
Terminal Domains

The AROM protein of Aspergillus nidulans is a multidomain pentafunctional polypeptide that is active as a dimer and catalyses steps 2-6 in the prechorismate section of the shikimate pathway. The three C-terminal domains (including the type I 3-dehydroquinase) of the AROM protein are homologous with the qutR-encoded QUTR protein that represses transcription of the eight genes comprising the quinic acid utilization (qut) gene cluster, and the two N-terminal domains are homologous with the qutA-encoded QUTA protein that transcribes the qut genes. As part of a larger research programme designed to compare the structures of the three proteins and to probe the domain structure and interaction within each protein, we have overproduced and purified the 3-dehydroquinase domain of the AROM protein. Additionally we have overproduced and purified the qutB-encoded quinate dehydrogenase and overproduced the qa-2 encoded type II 3-dehydroquinase of Neurospora crassa. We report that the AROM 3-dehydroquinase domain has a monomeric native state, with an apparent kcat./Km ratio that is approx. 160-fold lower than the value for the native N. crassa AROM protein. The AROM protein 3-dehydroquinase domain is sensitive to inactivation by borohydride in the presence of the substrate 3-dehydroquinate, confirming that it is a typical type I 3-dehydroquinase. The purified quinate dehydrogenase is bifunctional, being able to metabolize shikimate as a substrate. The apparent Km values for quinate (450 microM), shikimate (1.7 mM) and NAD+ (150 microM) are all similar to values reported for the qa-3-encoded enzyme from N. crassa.

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