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

Abstract

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.

Avirulent Isolates of Penicillium chrysogenum to Control the Blue Mold of Apple Caused by P. expansum

“…The two newly discovered P. chrysogenum isolates examined in this study were obtained from hygromycin-amended media in the laboratory. Hygromycin resistance has been attributed to the presence of two aminoglycoside phosphotransferases (APHs), specifically APH(4)-Ia and APH(7 )-Ia [43,44], and is often exploited as a genetic tool for fungal selection using the Agrobacterium tumefaciens-mediated transformation or as a dominant selectable marker via protoplast gene transformation studies [52]. Oddly enough, the two APH-encoding genes were not found in either the 404 or 413 genomes.…”

“…All other antimicrobial compounds inhibited the growth of all the Penicillium isolates tested (Table S3), showing that the isolates did not have a multidrug-resistant phenotype. To understand the basis of the hygromycin resistance in the two P. chrysogenum isolates, their genomic data were used to search for the homologs of previously identified hygromycin-resistant genes encoding two aminoglycoside phosphotransferases (APHs), specifically APH(4)-Ia and APH(7 )-Ia [43,44]. However, both genes encoding APH(4)-Ia and APH (7 ) were absent in each strain.…”

“…To understand the basis of the hygromycin resistance in the two P. chrysogenum isolates, their genomic data were used to search for the homologs of previously identified hygromycin-resistant genes encoding two aminoglycoside phosphotransferases (APHs), specifically APH(4)-Ia and APH(7″)-Ia [43,44]. However, both genes encoding APH(4)-Ia and APH(7″) were absent in each strain.…”

SAS: Split Antibiotic Selection for identifying chaperones that improve protein solubility

“…The N-terminal lobe contains the nucleotide binding domain where the C-terminal lobe binds to hygromycin B and contains the catalytic residues (Fig. 1B) [37]. To link protein solubility to hygromycin B resistance a site had to be selected within APH(7″) where a protein of interest would be inserted.…”