Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant

Iino, D.; Takakura, Yasuaki; Fukano, K.; Sasaki, Yasuyuki; Hoshino, Takayuki; Ohsawa, Kanju; Nakamura, Akira; Yajima, Seishi

doi:10.1016/j.jsb.2013.05.023

Cited by 13 publications

(32 citation statements)

References 34 publications

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“…The active site residues Ala95 and Asn203 form hydrogen bond, whereas Val47, Leu205, and Ile215 form non‐bonded contacts with proper intermolecular distances (Figure A and Table ). Earlier, it has been observed that the residues Arg49 and Asp60 form a salt bridge at a distance of 2.9 Å . However, binding of ZINC71575479 at NTP‐binding site of 3W0O lowered the salt bridge distance to 1.829 Å (Figure A and Table ).…”

Section: Resultsmentioning

confidence: 85%

“…(Table ) Crystal structure 3TDV is an APH2″ (IIIa) type of AK and has experimental Ki value for tyrphostin AG1478 as 59.3 µM . Complex structure of Hph5/APH(4)‐Ia with hygromycin and ATP analog (ADP or AMP‐PNP) from E. coli (3W0O) showed involvement of critical residues at nucleotide binding site . It has been identified that the catalytic site of Hph5 (3W0O) complexed with AMP‐PNP include residues such as Ala95, Arg93, Val47, Arg49, Leu205, Asn203, Ile215, and Asp216 .…”

Section: Resultsmentioning

confidence: 99%

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Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains

Parulekar

Sonawane

2017

J of Cellular Biochemistry

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Antibiotic resistance to aminoglycoside group of antibiotics mainly occurs by aminoglycoside kinases (AKs). Thus, targeting AKs from different multidrug resistant (MDR) strains could result into inhibition of AK enzymes and ultimately the resistance. Therefore, the present study aims to target one of these AKs that is APH(3')-Ia from Acinetobacter baumannii through structure based virtual screening (SBVS) and test the binding affinity of the most stable virtually screened inhibitor with AKs from Mycobacterium tuberculosis, Acinetobacter baumannii, Enterococcus gallinarum, and Escherichia coli. SBVS investigated ZINC71575479 as a most stable inhibitor with -8.92 kcal/mol of binding energy and 0.66 µM of inhibition constant. Molecular docking results revealed that the ZINC71575479 can efficiently bind to nucleotide triphosphate (NTP) binding site of different AKs which is a known drug target site. Sequence analysis study of different AKs showed no significant similarity for active site residues; however structure superimposition study showed conserved NTP-binding domain. Molecular dynamics (MD) simulations showed stable behavior of all docked complexes with notable conformational stability of salt bridges at NTP-binding site of different AKs. Binding energy calculations revealed the interactions between key residues from NTP- binding domain of different AKs with ZINC71575479. In order to validate the MD simulations and binding energy results, crystal structure complexed with tyrphostin AG1478 a known inhibitor of AKs was kept as control. Thus, this work demonstrates the binding efficiency of ZINC71575479 toward different AKs for circumventing aminoglycoside resistance and opens avenues for the development of new antibiotics that can target diverse MDR strains with aminoglycoside resistance.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains

Parulekar

Sonawane

2017

J of Cellular Biochemistry

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“…The mutant gene obtained, hph5, was functional as a selection marker in T. thermophilus at up to 65 C. 6) We have also reported that mutant protein HPH5 was thermostabilized at the enzyme level at 17 C, 7) as well as crystallization and structural analysis of it. 8,9) Recently, another thermostable hph mutant was found to be functional at up to 82 C in T. thermophilus, 10) but no precise enzymatic characterization of this mutant protein has been reported.…”

Section: )mentioning

confidence: 99%

Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase fromStreptomyces hygroscopicus

Sugimoto¹,

Takakura²,

Shiraki³

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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“…The APH family, represented by serine/threonine and tyrosine protein kinases in eukaryotes, contains Brenner's motif (HxDxxxxN;Brenner, 1978) as a conserved catalytic motif. The Asp residue in the motif transfers the phosphate group (Madhusudan et al, 1994;Cole et al, 1995;Hon et al, 1997;Iino et al, 2013). To investigate their substrate-recognition mechanisms and to exploit drug design, crystal structures of APHs have been determined.…”

Section: Introductionmentioning

confidence: 99%

“…It is not currently used for clinical purposes, but it is widely used as a tool in the cloning of genes for both prokaryotes and eukaryotes in molecular-biology experiments (Addgene; http://www.addgene.org). To elucidate the inactivation mechanism of HygB, the crystal structure of APH(4)-Ia from Escherichia coli in a ternary complex with HygB and 5 0 -adenylyl-,-imidodiphosphate (AMP-PNP) has been reported (Stogios et al, 2011;Iino et al, 2013). In this structure, the hydroxyl O atom at position 4 in the hyosamine ring was located between Asp198 in Brenner's motif and the -phosphate of AMP-PNP.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus

et al. 2019

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Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular‐biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′‐phosphotransferase‐Ia [APH(7′′)‐Ia] from Streptomyces hygroscopicus and aminoglycoside 4‐phosphotransferase‐Ia [APH(4)‐Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)‐Ia was reported as a ternary complex with HygB and 5′‐adenylyl‐β,γ‐imidodiphosphate (AMP‐PNP). To investigate the differences in the substrate‐recognition mechanism between APH(7′′)‐Ia and APH(4)‐Ia, the crystal structure of APH(7′′)‐Ia complexed with HygB is reported. The overall structure of APH(7′′)‐Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)‐Ia, and consists of an N‐terminal lobe (N‐lobe) and a C‐terminal lobe (C‐lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)‐Ia and APH(4)‐Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C‐lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)‐Ia and APH(4)‐Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.

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Crystal structures of the ternary complex of APH(4)-Ia/Hph with hygromycin B and an ATP analog using a thermostable mutant

Cited by 13 publications

References 34 publications

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains

Molecular modeling studies to explore the binding affinity of virtually screened inhibitor toward different aminoglycoside kinases from diverse MDR strains

Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase fromStreptomyces hygroscopicus

Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus

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