A kinase bioscavenger provides antibiotic resistance by extremely tight substrate binding

Microbial communities are self-controlled by repertoires of lethal agents, the antibiotics. In their turn, these antibiotics are regulated by bioscavengers that are selected in the course of evolution. Kinase-mediated phosphorylation represents one of the general strategies for the emergence of antibiotic resistance. A new subfamily of AmiN-like kinases, isolated from the Siberian bear microbiome, inactivates antibiotic amicoumacin by phosphorylation. The nanomolar substrate affinity defines AmiN as a phosphotransferase with a unique catalytic efficiency proximal to the diffusion limit. Crystallographic analysis and multiscale simulations revealed a catalytically perfect mechanism providing phosphorylation exclusively in the case of a closed active site that counteracts substrate promiscuity. AmiN kinase is a member of the previously unknown subfamily representing the first evidence of a specialized phosphotransferase bioscavenger.

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Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽

10.3390/biom11121882 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1882

Author(s):

Wei Xia ◽

Yingguo Bai ◽

Pengjun Shi

Keyword(s):

Hydrogen Bonding ◽

Rational Design ◽

Substrate Binding ◽

Enzymatic Saccharification ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Glycoside Hydrolases ◽

Cellulosic Biomass ◽

Substrate Affinity ◽

Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

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Crystallographic analysis shows substrate binding at the −3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases

Biochemical Journal ◽

10.1042/bj20071128 ◽

2008 ◽

Vol 410 (1) ◽

pp. 71-79 ◽

Cited By ~ 51

Author(s):

Elien Vandermarliere ◽

Tine M. Bourgois ◽

Sigrid Rombouts ◽

Steven van Campenhout ◽

Guido Volckaert ◽

...

Keyword(s):

Binding Site ◽

Active Site ◽

Glycoside Hydrolase ◽

Plant Cell Wall ◽

Substrate Binding ◽

Crystallographic Analysis ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules ◽

Hydrolase Family

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein–ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the −1 and −2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the −3 and +1 subsite showing the spanning of the −1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.

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EPR Study of Substrate Binding to the Mn(II) Active Site of the Bacterial Antibiotic Resistance Enzyme FosA: A Better Way To Examine Mn(II)

Journal of the American Chemical Society ◽

10.1021/ja012480f ◽

2002 ◽

Vol 124 (10) ◽

pp. 2318-2326 ◽

Cited By ~ 37

Author(s):

Stoyan K. Smoukov ◽

Joshua Telser ◽

Bryan A. Bernat ◽

Chris L. Rife ◽

Richard N. Armstrong ◽

...

Keyword(s):

Antibiotic Resistance ◽

Active Site ◽

Substrate Binding ◽

Bacterial Antibiotic Resistance

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ω-Oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site

Biochemical Journal ◽

10.1042/bj3360345 ◽

1998 ◽

Vol 336 (2) ◽

pp. 345-352 ◽

Cited By ~ 15

Author(s):

Igor IVANOV ◽

Kristin SCHWARZ ◽

Herman G. HOLZHÜTTER ◽

Galina MYAGKOVA ◽

Hartmut KÜHN

Keyword(s):

Fatty Acids ◽

Active Site ◽

Substrate Binding ◽

Salt Bridge ◽

Binding Pocket ◽

Substrate Affinity ◽

Carboxylate Group ◽

Fatty Acid Binding ◽

Substrate Orientation ◽

Binding Cleft

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the ω-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of ω-modified fatty acids by 15-lipoxygenases and found that ω-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, ω-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403 → Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of ω-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the ω-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases.

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Role of lysine, tryptophan and calcium in the β-elimination activity of a low-molecular-mass pectate lyase from Fusarium moniliformae

Biochemical Journal ◽

10.1042/bj3190159 ◽

1996 ◽

Vol 319 (1) ◽

pp. 159-164 ◽

Cited By ~ 25

Author(s):

M. Narsimha RAO ◽

Asha A. KEMBHAVI ◽

Aditi PANT

Keyword(s):

Fluorescence Quenching ◽

Molecular Mass ◽

Active Site ◽

Structural Changes ◽

Substrate Binding ◽

Gel Filtration ◽

Pectate Lyase ◽

Tryptophan Fluorescence ◽

Emission Spectrometry ◽

Substrate Affinity

An extracellular pectate lyase from Fusarium moniliformae was purified to homogeneity by affinity chromatography followed by gel filtration, with a yield of 76.5%. Laser desorption MS of the enzyme gave a molecular mass of 12133.5±2.5 Da. The pectate lyase was a glycoprotein with a 5% carbohydrate content and had a pI value of 9.1. Atomic-emission spectrometry showed that Ca2+ was a part of the holoenzyme held by carboxy groups of the protein. These results support the hypothesis of a putative Ca2+ site suggested by Yodder, Keen and Jurnak [(1993) Science 260, 1503–1507] in the crystal structure of pectate lyase C of Erwinia chrysanthemi. Loss of Ca2+ was observed by treatment with EGTA or carboxy-modifying Woodward's reagent K, with subsequent loss of enzyme activity. Tryptophan fluorescence quenching showed that Ca2+ does not affect binding of substrate to enzyme. Chemical-modification and substrate-protection studies showed the presence of lysine and tryptophan at or near the active site of the pectate lyase. Chemically modified enzyme showed no major structural changes as determined by CD. Amino acid analyses of native, trinitrobenzenesulphonate (TBNS)-treated and substrate-protected TNBS-treated enzyme showed that a single essential residue of lysine is present at or near the active-site. Substrate-affinity studies showed that tryptophan could be essential for substrate binding, whereas lysine could be involved in the catalysis. Fluorescence quenching further confirmed the involvement of tryptophan in substrate binding. The reaction mechanism involving β-elimination by this enzyme is discussed.

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