Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase

2001 ◽  
Vol 47 (10) ◽  
pp. 961-964 ◽  
Author(s):  
Paxton Loke ◽  
Tiow-Suan Sim
Keyword(s):  
Mutational Analysis ◽  
Soluble Expression ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cephalosporium Acremonium ◽  
Multiple Sequence ◽  
Local Protein Structure ◽  
Reduced Temperature ◽  
Isopenicillin N ◽  
Local Protein

Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of δ -(L-α-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37°C. Soluble expression was restored at a reduced temperature of 25°C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.Key words: isopenicillin N synthase, site-directed mutagenesis, glycine, Cephalosporium acremonium.

Site-Directed Mutagenesis of Proline-285 to Leucine in Cephalosporium acremonium Isopenicillin N - Synthase Affects Catalysis and Increases Soluble Expression at Higher Temperatures

2001 ◽  
Vol 56 (5-6) ◽  
pp. 413-415 ◽  
Author(s):  
Paxton Loke ◽  
Tiow-Suan Sim
Keyword(s):  
Biosynthetic Pathway ◽  
Soluble Expression ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cephalosporium Acremonium ◽  
E Coli ◽  
Measurable Activity ◽  
Important Enzyme ◽  
Isopenicillin N

The conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine (ACV) to isopenicillin N is dependant on the catalytic action of isopenicillin N - synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. One of the amino acid residues suggested by the Aspergillus nidulans IPNS crystal structure for interaction with the valine isopropyl group of ACV is proline-283. Site-directed mutagenesis of the corresponding proline- 285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher temperatures in a heterologous E. coli host.

Mutational analysis of transmembrane histidines in the amiloride-sensitive Na+/H+ exchanger

1995 ◽  
Vol 269 (2) ◽  
pp. C392-C402 ◽  
Author(s):  
D. Wang ◽  
D. F. Balkovetz ◽  
D. G. Warnock
Keyword(s):  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
Clear Cut ◽  
Inhibition Concentration ◽  
Nhe1 Activity ◽  
Per Se ◽  
Nhe Isoforms

The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the human amiloride-sensitive Na+/H+ exchanger (NHE1) in stably transfected fibroblasts. NHE1 was protected by cimetidine and amiloride from DEPC, and DEPC inhibition was reversed with hydroxylamine, suggesting a role for critical histidine groups in NHE activity. We replaced the histidines (H) in putative transmembrane domains (H35, H120, H349) with glycine (G) using site-directed mutagenesis. There was no significant change in NHE activity of the H120G; H349G; H120,349G; and H35,120,349G mutants compared with wild type. The 50% inhibition concentration values for amiloride, ethyl isopropyl amiloride (EIPA), and cimetidine of the H349G mutant were significantly increased compared with the wild-type NHE1. We also examined the DEPC effect on the transport activity of the triple histidine mutant (H35,120,349G) and found that NHE1 activity was still inhibited by DEPC with reversal by hydroxylamine and protected by amiloride and cimetidine. Kinetic analysis of DEPC inhibition indicated that two "critical" histidine residues are required for NHE transport activity. Substitutions of H349 with asparagine (N), glutamine (Q), serine (S), tyrosine (Y), valine (V), leucine (L), and phenylalanine (F) were also examined. There were no changes in NHE activity of these mutants compared with wild type. The H349G and H349L mutants became more resistant to amiloride, whereas the H349Y and H349F mutants became more sensitive to amiloride. The H349S (mimics NHE3) and H349Y (mimics NHE4) mutations had only modest effects on amiloride sensitivity. These results indicate that H349 affects the interaction of NHE1 with its inhibitors, even though substitutions at this site, per se, do not appear to explain the differences in amiloride sensitivity between different NHE isoforms. Despite clear-cut effects of the H349G mutation on the competitive interaction of NHE1 with cimetidine and EIPA, this mutation did not affect the affinity of NHE1 for its cationic substrates (Na+, Li+).

scholarly journals Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

2005 ◽  
Vol 71 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Chan K. Chan Kwo Chion ◽  
Sarah E. Askew ◽  
David J. Leak
Keyword(s):  
Mutational Analysis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Styrene Oxidation ◽  
Overlapping Reading Frames ◽  
Reading Frames

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.

scholarly journals Mutational Analysis of Xanthomonas Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants

2004 ◽  
Vol 186 (18) ◽  
pp. 6239-6247 ◽  
Author(s):  
Jung-Gun Kim ◽  
Eunkyung Jeon ◽  
Jonghee Oh ◽  
Jae Sun Moon ◽  
Ingyu Hwang
Keyword(s):  
Amino Acid ◽  
Hypersensitive Response ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Tobacco Plants ◽  
Functional Region ◽  
Amino Acid Peptide ◽  
Α Helix

ABSTRACT HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp. The secondary structure of the peptide is predicted to be an α-helix, as is the HrpW region that is homologous to HpaG. The predicted α-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a xopA gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.

scholarly journals Rapid Oligonucleotide-Based Recombineering of the Chromosome of Salmonella enterica

2009 ◽  
Vol 75 (6) ◽  
pp. 1575-1580 ◽  
Author(s):  
Roman G. Gerlach ◽  
Daniela Jäckel ◽  
Stefanie U. Hölzer ◽  
Michael Hensel
Keyword(s):  
Salmonella Enterica ◽  
Mutational Analysis ◽  
Bacterial Species ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Synthetic Dna ◽  
Chromosomal Genes ◽  
Bacterial Chromosomes ◽  
Chromosomal Loci

ABSTRACT Recombinant engineering using Red recombinase-based approaches offers efficient and rapid approaches to deletion and modification of genes. Here we describe a novel application of Red recombinant engineering that enables direct manipulation of chromosomal loci by electroporation with short synthetic DNA molecules. We demonstrate the use of this approach for the generation of scarless in-frame deletions in chromosomal genes of Salmonella enterica. Furthermore, we describe rapid site-directed mutagenesis within bacterial chromosomes without any requirement for cloning and mutating genes in vitro or for reintroducing mutant alleles into the chromosome. This approach can be expected to facilitate mutational analysis in S. enterica and in other bacterial species able to support Red-mediated recombination.

scholarly journals Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

2006 ◽  
Vol 188 (17) ◽  
pp. 6179-6183 ◽  
Author(s):  
Jung-Kul Lee ◽  
Ee-Lui Ang ◽  
Huimin Zhao
Keyword(s):  
Substrate Specificity ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Enzyme Substrate ◽  
Molecular Determinants

ABSTRACT Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

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