Substrate specificity of Nudix hydrolases from &lt;i&gt;Myxococcus xanthus&lt;/i&gt;

Kimura, Yoshinobu; Yamamoto, Yuuka; Kajimoto, Sayaka; Sakai, Ai; Takegawa, Kaoru

doi:10.2323/jgam.2017.07.001

Cited by 2 publications

(4 citation statements)

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“…50 However, Mn 2+ is required for the growth and survival of bacteria, and the intracellular Mn 2+ levels are homeostatically regulated. 51 Several of M. xanthus enzymes that we have studied are activated by Mn 2+ but not by Mg 2+ (e.g., protein phosphatases, acetyl-CoA synthase, aminoacyl-tRNA synthetases, and Nudix hydrolases), [52][53][54][55][56][57][58] suggesting that the concentration of intracellular Mn 2+ in M. xanthus should be sufficient to support catalytic activity.…”

Section: Gsh Synthesis By Mxgcl2 and Mxgsmentioning

confidence: 95%

Characterization of glutamate‐cysteine ligase and glutathione synthetase from the δ‐proteobacterium Myxococcus xanthus

Okada

Kimura

2022

Proteins

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Glutathione (GSH) is synthesized in two ATP-dependent reactions by glutamatecysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gramnegative bacterium belonging to δ-proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively.MxGcl2 was activated by Mn 2+ , but not by Mg 2+ , and stabilized in the presence of 5 mM Mn 2+ or Mg 2+ . Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with K i = 2.1 μM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg 2+ but strongly inhibited by Mn 2+ even in the presence of 10 mM Mg 2+ . MxGs was inhibited by GSSG at K i = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.

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Section: Gsh Synthesis By Mxgcl2 and Mxgsmentioning

confidence: 95%

Characterization of glutamate‐cysteine ligase and glutathione synthetase from the δ‐proteobacterium Myxococcus xanthus

Okada

Kimura

2022

Proteins

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“…Many Nudix hydrolases require Mg 2+ for maximum activity, and Mn 2+ or Zn 2+ for partial activity. We have previously reported that M. xanthus Nudix hydrolases for Ap 4 A were stimulated by Mn 2+ , Mg 2+ , or Co 2+ (Kimura et al, 2018). When Nud2 activities for 13 substrates (four each of oxidized purine deoxynucleotides, unoxidized purine deoxynucleotides, and unoxidized purine nucleotides, and a diadenosine triphosphate (Ap 4 A)) were measured in the presence of 5 mM each of Mn 2+ , Mg 2+ , or Co 2+ , Mn 2+ was found to stimulate all the activities (Fig.…”

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confidence: 98%

“…In bacteria, the number of Nudix genes varies from 0 to >30 (McLennan, 2006). We have previously reported that Myxococcus xanthus contains 12 Nudix hydrolases (Kimura et al, 2018). Among them, 11 His-tagged Nudix hydrolases (Nud1-Nud11) have been expressed in E. coli, and their substrate specificities have been studied (Kimura et al, 2018).…”

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confidence: 99%

“…We have previously reported that Myxococcus xanthus contains 12 Nudix hydrolases (Kimura et al, 2018). Among them, 11 His-tagged Nudix hydrolases (Nud1-Nud11) have been expressed in E. coli, and their substrate specificities have been studied (Kimura et al, 2018). Nud2 (MXAN_1246) showed high hydrolase activity towards Ap 4 A, Ap 5 A, ATP, ADP, GTP, GDP, and especially 8-oxo-dGTP.…”

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Enzymatic characteristics of Nudix hydrolase 2 (Nud2), an 8-oxo-dGTP hydrolase from <i>Myxococcus xanthus</i>

Kimura

Kajimoto

Yamamoto

et al. 2020

J. Gen. Appl. Microbiol.

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Myxococcus xanthusNudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn 2+ was the most effective cofactor for stimulating oxidized deoxynucleotide hydrolase activity. The K m of Nud2 with 8-oxo-dGTP for Mn 2+ was 19-fold lower than that for Mg 2+ , and was 2-fold lower than that with dGTP for Mn 2+ . The specificity constant (k cat /K m ) for 8oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif ( 84 AX 5 90 GX 7 REX 2 EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.Nudix hydrolases are a family of pyrophosphatases containing the highly conserved 23-residue sequence segment (GX 5 EX 7 REUXEEXGU, where U is a bulky hydrophobic amino acid such as Ile, Leu, or Val, and X is any amino acid) called the Nudix motif . They are metal-dependent enzymes found in all classes of organism, and can hydrolyze a wide range of organic pyrophosphates, including nucleoside di-and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars, RNA caps, NAD(P)H, coenzyme A, and guanosine-3¢,5¢-tetraphosphate (ppGpp) with varying degrees of substrate specificity (McLennan, 2006). As these substrates are involved in energy metabo-8-oxo-dGTP hydrolase of M. xanthus

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Substrate specificity of Nudix hydrolases from <i>Myxococcus xanthus</i>

Cited by 2 publications

References 21 publications

Characterization of glutamate‐cysteine ligase and glutathione synthetase from the δ‐proteobacterium Myxococcus xanthus

Characterization of glutamate‐cysteine ligase and glutathione synthetase from the δ‐proteobacterium Myxococcus xanthus

Enzymatic characteristics of Nudix hydrolase 2 (Nud2), an 8-oxo-dGTP hydrolase from <i>Myxococcus xanthus</i>

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