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The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

Alperstein A, Ulrich B, Garofalo DM, Dreisbach R, Raff H, Sheppard K - PLoS ONE (2014)

Bottom Line: Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn.This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner.Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America.

ABSTRACT
The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

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B. bacteriovorus AspRS aspartylates tRNAAsn.Aminoacylation of in vitro transcribed tRNAAsp (○) and tRNAAsn (▵) by either (A) B. bacteriovorus AspRS or (B) B. bacteriovorus AsnRS. Reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNAAsp or Asn, 11.0 µM tRNAAsp or Asn, 4.0 mM ATP, 4.0 mM relevant amino acid (L-Asp or L-Asn) and 3.0 µM enzyme. Experiments were repeated three times and error bars represent standard deviations.
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pone-0110842-g001: B. bacteriovorus AspRS aspartylates tRNAAsn.Aminoacylation of in vitro transcribed tRNAAsp (○) and tRNAAsn (▵) by either (A) B. bacteriovorus AspRS or (B) B. bacteriovorus AsnRS. Reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNAAsp or Asn, 11.0 µM tRNAAsp or Asn, 4.0 mM ATP, 4.0 mM relevant amino acid (L-Asp or L-Asn) and 3.0 µM enzyme. Experiments were repeated three times and error bars represent standard deviations.

Mentions: To determine if the B. bacteriovorus AspRS is non-discriminating, we overproduced the enzyme in E. coli and purified it to homogeneity [24]. The recombinant enzyme was readily able to aspartylate both tRNAAsp and tRNAAsn to similar levels (Figure 1A). Discriminating AspRS enzymes typically prefer tRNAAsp to tRNAAsn by a factor of 500–2,250 [22], [37]. In contrast and similar to other ND-AspRS enzymes [22], [24], [37], [38], the B. bacteriovorus AspRS preferred tRNAAsp as a substrate by only 3-fold (Table 1). The difference in catalytic efficiency by AspRS was attributed to an increased kcat with tRNAAsp as a substrate (Table 1). The B. bacteriovorus AsnRS also readily uses tRNAAsn as a substrate, reaching a similar aminoacylation plateau (Figure 1B). The tRNAAsn was a better substrate for AsnRS by 3-fold with a higher kcat compensating for an increased KM relative to AspRS (Table 1).


The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

Alperstein A, Ulrich B, Garofalo DM, Dreisbach R, Raff H, Sheppard K - PLoS ONE (2014)

B. bacteriovorus AspRS aspartylates tRNAAsn.Aminoacylation of in vitro transcribed tRNAAsp (○) and tRNAAsn (▵) by either (A) B. bacteriovorus AspRS or (B) B. bacteriovorus AsnRS. Reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNAAsp or Asn, 11.0 µM tRNAAsp or Asn, 4.0 mM ATP, 4.0 mM relevant amino acid (L-Asp or L-Asn) and 3.0 µM enzyme. Experiments were repeated three times and error bars represent standard deviations.
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Related In: Results  -  Collection

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pone-0110842-g001: B. bacteriovorus AspRS aspartylates tRNAAsn.Aminoacylation of in vitro transcribed tRNAAsp (○) and tRNAAsn (▵) by either (A) B. bacteriovorus AspRS or (B) B. bacteriovorus AsnRS. Reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNAAsp or Asn, 11.0 µM tRNAAsp or Asn, 4.0 mM ATP, 4.0 mM relevant amino acid (L-Asp or L-Asn) and 3.0 µM enzyme. Experiments were repeated three times and error bars represent standard deviations.
Mentions: To determine if the B. bacteriovorus AspRS is non-discriminating, we overproduced the enzyme in E. coli and purified it to homogeneity [24]. The recombinant enzyme was readily able to aspartylate both tRNAAsp and tRNAAsn to similar levels (Figure 1A). Discriminating AspRS enzymes typically prefer tRNAAsp to tRNAAsn by a factor of 500–2,250 [22], [37]. In contrast and similar to other ND-AspRS enzymes [22], [24], [37], [38], the B. bacteriovorus AspRS preferred tRNAAsp as a substrate by only 3-fold (Table 1). The difference in catalytic efficiency by AspRS was attributed to an increased kcat with tRNAAsp as a substrate (Table 1). The B. bacteriovorus AsnRS also readily uses tRNAAsn as a substrate, reaching a similar aminoacylation plateau (Figure 1B). The tRNAAsn was a better substrate for AsnRS by 3-fold with a higher kcat compensating for an increased KM relative to AspRS (Table 1).

Bottom Line: Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn.This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner.Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America.

ABSTRACT
The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

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