High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Zhang, Lian-Hui; Xu, Jiahao; Birch, Robert G.

doi:10.1099/00221287-144-2-555

Cited by 30 publications

(55 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…albA in pGST-AlbA was PCR-amplified from pJP1076, which was used in the original sequencing of the albA gene from K. oxytoca JMP 4505 (Walker et al, 1988;Zhang et al, 1998b). Therefore, we cloned albA from K. oxytoca ATCC 13182 T and confirmed the sequence using clones from five separate PCR amplifications.…”

Section: Resultsmentioning

confidence: 99%

“…AlbA and its variants were expressed separately as glutathione S-transferase (GST) fusion proteins, from which the recombinant AlbA and the derivatives were released by incubation with thrombin. After confirmation of their purity using SDS-PAGE, the proteins were freeze-dried and stored at 220˚C (Zhang et al, 1998b). The protein concentration was determined by means of the DC protein assay (Bio-Rad) with BSA as the standard.…”

Section: Methodsmentioning

confidence: 99%

“…AlbA variants (0?77 mM) in TMM buffer (10 mM Tris/HCl, pH 7?0; 10 mM MgCl 2 ; 2 mM 2-mercaptoethanol) were mixed with albicidin at concentrations ranging from 0?12 to 1?17 mM, then incubated at 25˚C for 5 min before a quantitative assay of free albicidin as described previously (Zhang et al, 1998b). Briefly, 100 ml of a fresh E. coli DH5a culture, which was adjusted to an OD 600 value of 1?8, were added to 5 ml of 1 % (w/v) agarose at 50˚C.…”

Section: Methodsmentioning

confidence: 99%

“…We used DEPC, a histidine-specific reagent, to probe the role of the histidine residues of AlbA in binding to albicidin, as a previous study had suggested that histidine residues could be involved in the protein-ligand interaction (Zhang et al, 1998b). After incubation of AlbA with 0?05 mM DEPC for 13 min, about 55 % of the albicidin-binding activity was lost.…”

Section: Effect Of Depc On Albicidin-binding Activity Of Albamentioning

confidence: 99%

“…This is of interest as a potential phytotoxin-and disease-resistance mechanism, and as a novel ligand-matrix combination for biotechnological applications (Walker et al, 1988;Zhang et al, 1998b). Kinetic and stoichiometric analyses indicate the presence of a single high-affinity binding site with a dissociation constant of 6?4610 28 M in the AlbA protein (Zhang et al, 1998b). Albicidin-binding capacity decreases by 30 % when pH is shifted from 6 to 4, which is the range for side-chain ionization of histidine residues.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Weng

et al. 2003

Microbiology

Self Cite

View full text Add to dashboard Cite

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30 %, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182 T to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His 78 , His 125 , His 141 and His 189 ) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1: 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His 125 might be the residue required for albicidin binding. Mutation of His 125 to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His 125 totally abolished binding activity. Mutation of His 125 to arginine and tyrosine had no effect. These results indicate that His 125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Effect Of Depc On Albicidin-binding Activity Of Albamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Weng

et al. 2003

Microbiology

Self Cite

View full text Add to dashboard Cite

show abstract

Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids

Testolin

Richter

Ritter

et al. 2022

Chemistry A European J

View full text Add to dashboard Cite

The rise of antibiotic resistance causes a serious health care problem, and its counterfeit demands novel, innovative concepts. The combination of photopharmacology, enabling a light-controlled reversible modulation of drug activity, with antibiotic drug design has led to first photoswitchable antibiotic compounds derived from established scaffolds. In this study, we converted cystobactamids, gyraseinhibiting natural products with an oligoaryl scaffold and highly potent antibacterial activities, into photoswitchable agents by inserting azobenzene in the N-terminal part and/or an acylhydrazone moiety near the C-terminus, yielding twenty analogs that contain mono-as well as double-switches. Antibiotic and gyrase inhibition properties could be modulated 3.4-fold and 5-fold by light, respectively. Notably, the sensitivity of photoswitchable cystobactamids towards two known resistance factors, the peptidase AlbD and the scavenger protein AlbA, was light-dependent. While irradiation of an analog with an N-terminal azobenzene with 365 nm light led to less degradation by AlbD, the AlbAmediated inactivation was induced. This provides a proof-ofprinciple that resistance towards photoswitchable antibiotics can be optically controlled.

show abstract

Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance

Weng

Hai-hua²,

et al. 2006

Pest Management Science

View full text Add to dashboard Cite

Five elite sugarcane breeding lines were tested for efficiency in embryogenesis and plant regeneration. All of them produced regenerative embryogenic calli but with varied efficiencies. To engineer strongly insect-resistant sugarcanes, the GC content of a truncated cry1Ac gene, which encodes the active region of Cry1Ac insecticidal delta-endotoxin, was increased from the original 37.4 to 47.5% following the sugarcane codon usage pattern. The synthetic cry1Ac gene (s-cry1Ac) was placed under the control of maize ubiquitin promoter and introduced by microprojectile bombardment into the embryogenic calli of sugarcane lines YT79-177 and ROC16. Southern blotting analysis showed that multicopies of s-cry1Ac were integrated into the genomes of transgenic sugarcane lines. Immunoblotting analysis identified 18 transgenic lines expressing detectable levels of s-Cry1Ac, which were estimated in the range of 1.8-10.0 ng mg(-1) total soluble proteins. Four transgenic and two parental lines were assayed for sugarcane stem borer resistance in leaf tissue feeding trials and greenhouse plant assays. The results showed that, while the untransformed control lines were severely damaged in both leaves and stems, the transgenic sugarcane lines expressing high levels of s-Cry1Ac proteins were highly resistant to sugarcane stem borer attack, resulting in complete mortality of the inoculated insects within 1 week after inoculation.

show abstract

High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Cited by 30 publications

References 18 publications

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids

Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance

Contact Info

Product

Resources

About