Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus

2006 ◽  
Vol 34 (1) ◽  
pp. 118-121 ◽  
Author(s):  
E.J. Dridge ◽  
D.J. Richardson ◽  
R.J. Lewis ◽  
C.S. Butler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Archaeoglobus Fulgidus ◽  
Substrate Selectivity ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
X Ray ◽  
Group Type

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Structure of the rat platelet factor 4 gene: a marker for megakaryocyte differentiation.

1987 ◽  
Vol 7 (2) ◽  
pp. 898-904 ◽  
Author(s):  
T Doi ◽  
S M Greenberg ◽  
R D Rosenberg
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Genomic Library ◽  
Transcriptional Start Site ◽  
Platelet Factor 4 ◽  
Start Codon ◽  
Cdna Expression Library ◽  
Amino Acid Residues ◽  
Platelet Factor ◽  
Nuclease Mapping

A rat platelet factor 4 (PF4) cDNA has been isolated by immunoscreening a g lambda 11 rat megakaryocyte cDNA expression library. Sequence analysis of the rat PF4 cDNA revealed that this megakaryocyte protein is composed of a leader sequence of 29 amino acid residues and a mature protein sequence of 76 amino acid residues. The structure of rat PF4 derived from its cDNA shows a marked homology with the amino acid sequence of human PF4 obtained by classical protein chemistry techniques. This observation is particularly striking with regard to the carboxy-terminal region of rat and human PF4, where 28 of the last 31 C-terminal residues are identical. The rat PF4 gene was obtained from a rat genomic library by using rat PF4 cDNA as a hybridization probe. Sequence analysis showed that the gene is constructed of three exons and two short introns. The transcriptional start site is located 73 base pairs upstream of the translational start codon as judged by S1 nuclease mapping and primer extension. The 5' noncoding region of the gene also exhibited a sequence homologous to the TATA box at -31, as well as a series of direct and inverted repeat sequences and a cluster of 26 T residues at -155 to -218. This latter domain may be involved in regulating PF4 gene expression during megakaryocytopoiesis.

Amino acid residues in asparaginase (Escherichia coli Hap) associated with its enzymic activity

1971 ◽  
Vol 227 (1) ◽  
pp. 171-179 ◽  
Author(s):  
Yoshihisa Nishimura ◽  
Hiroshi Makino ◽  
Osamu Takenaka ◽  
Yuji Inada
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Amino Acid Residues

scholarly journals Diarrhea-Inducing Activity of Avian Rotavirus NSP4 Glycoproteins, Which Differ Greatly from Mammalian Rotavirus NSP4 Glycoproteins in Deduced Amino Acid Sequence, in Suckling Mice

2002 ◽  
Vol 76 (11) ◽  
pp. 5829-5834 ◽  
Author(s):  
Yoshio Mori ◽  
Mohammed Ali Borgan ◽  
Naoto Ito ◽  
Makoto Sugiyama ◽  
Nobuyuki Minamoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Suckling Mice

ABSTRACT Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences. The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4.

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7913-7924
Author(s):  
J R Geiser ◽  
H A Sundberg ◽  
B H Chang ◽  
E G Muller ◽  
T N Davis
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Binding Site ◽  
Hybrid System ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Acid Residues ◽  
Calmodulin Binding ◽  
Pole Body ◽  
Two Hybrid

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.

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