Identification of catalytically essential amino acid residues of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105

ABSTRACT A tag of three lysines alternating with three glycines was added to the C-terminal end of the β chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.

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Cloning, Overexpression, and Characterization of a Novel Thermostable Penicillin G Acylase from Achromobacter xylosoxidans: Probing the Molecular Basis for Its High Thermostability

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2764-2770.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2764-2770 ◽

Cited By ~ 30

Author(s):

Gang Cai ◽

Songcheng Zhu ◽

Sheng Yang ◽

Guoping Zhao ◽

Weihong Jiang

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Molecular Basis ◽

Alcaligenes Faecalis ◽

Amino Acid Identity ◽

Penicillin G Acylase ◽

Penicillin G ◽

Achromobacter Xylosoxidans ◽

High Thermostability ◽

Arginine Residues

ABSTRACT The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2,586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60°C and its half-life of inactivation at 55°C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.

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Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coli. Relation to the orientation of membrane proteins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)77691-1 ◽

1988 ◽

Vol 263 (36) ◽

pp. 19690-19696

Author(s):

K Yamane ◽

S Mizushima

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Membrane Proteins ◽

Signal Peptide ◽

Cytoplasmic Membrane ◽

Protein Translocation ◽

Basic Amino Acid ◽

Amino Acid Residues

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Alterations of the carboxyl-terminal amino acid residues of Escherichia coli lipoprotein affect the formation of murein-bound lipoprotein.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)41811-x ◽

1992 ◽

Vol 267 (27) ◽

pp. 19560-19564

Author(s):

W.Y. Zhang ◽

H.C. Wu

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Carboxyl Terminal

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

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0030105 ◽

1989 ◽

Vol 3 (2) ◽

pp. 105-112 ◽

Cited By ~ 5

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.

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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

Biochemical Society Transactions ◽

10.1042/bst0340118 ◽

2006 ◽

Vol 34 (1) ◽

pp. 118-121 ◽

Cited By ~ 9

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.

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