Aminoacyl-transferase

Honjo, J., Nishizuka, Y., Hayaishi, O., and Kato, I. (1968) Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J. Biol. Cbem. 243, 3553-3555. 

The aminoacyl transfer reaction, one of the latter stages in protein synthesis, involves incorporation of amino acids from soluble ribonucleic acid-amino acid into ribosomal protein. This reaction requires guanosine triphosphate and a soluble portion of the cell. Evidence has been obtained with rat liver preparations that aminoacyl transfer is catalyzed by two protein factors, aminoacyl transferases (or polymerases) I and n, which have been resolved and partially purified from the soluble fraction. Transferase n activity has also been obtained from deoxycholate-soluble extracts of microsomes. With purified transferases I and n, incorporation is observed with relatively low levels of GTP its sulfhy-dryl requirement is met by a variety of compounds. The characteristics of this purified amino acid incorporating system, in terms of dependency on the concentration of its components, are described. 

Aminoacyl transferase I represents a highly purified (500- to 1000-fold) preparation of the initial 35 to 60% A.S. residue described above, obtained from the pH 5 Supernatant by further fractionation with ammo-... 

The transfer of labeled amino acids from aminoacyl sRNA to purified rat-liver ribonucleoprotein particles has been shown to require GTP, and a soluble portion (pH 5 Supernatant) of the cell. An enzyme fraction, aminoacyl transferase (or polymerase) I, purified from the pH 5 Supernatant was found to catalyze the transfer of amino acid to protein with microsomes, but not with the more purified ribonucleoprotein particles (ribosomes). When transferase I was supplemented with glutathione and a microsomal extract, microsomal aminoacyl transferase (or polymerase) H, transferring activity was restored. Since the pH 5 Supernatant was active in catalyzing the transfer of amino acids from sRNA to ribosomal protein, it was concluded that both transferring activities were present in this crude fraction. Resolution of the two activities from the pH 5 Supernatant fraction was obtained by salt-fractionation procedures. Neither enzyme fraction was active when incubated individually or with glutathione, but together in the presence of... 

Figure 13 General substrate specificity assays, (a) Adenylation domain, (b) Condensation/TGH domain, (c) Tailoring enzyme, for example, O-methyltransferase. (d) Aminoacyl transferase.

Adenylation domains utilize free amino acids,97 aryl acids,104 or fatty acids,51 biosynthetic substrates, and one T domain for tethering their substrates on the thiotemplate. C domains have an upstream nucleophile and a downstream electrophile as biosynthetic substrates, and two T domain substrates one upstream and one downstream T domain.93 10 Transferases, such as the aminoacyl transferase CmaE in the crotonine biosynthetic pathway, 6 are similar to C domains in that they use two T domain substrates but they have only one biosynthetic substrate tethered to the downstream T domain. Tailoring enzymes can have two forms of biosynthetic substrates T domain-bound substrates52 5 or non-T domain-bound substrates. Non-T domain-bound substrates can be free carbon acids,46 107 CoA-activated species, or the analogue of the natural product lacking the assayed chemical modification. 

Aminoacyl transferases can be characterized in their native biosynthetic substrate by tethering the predicted biosynthetic substrate on the downstream T domain and MS detection of substrate transfer to the upstream T domain upon incubation with the transferase. Biosynthetic substrate tolerance of transferases is tested by loading biosynthetic substrates differing from the native substrate on the downstream T domain and by the same approach as for substrate identification. 

MS-based assays that are aimed to characterize the specificity of catalytic NRPS domains and tailoring enzymes for carrier protein substrates can be done on high-resolution mass spectrometers or, for small substrate T domains (<20 kDa),105 on low-resolution mass spectrometers. For investigation of T domain substrate tolerance, the native T domain substrates of a catalytic NRPS domain or tailoring enzyme are exchanged by different T domains, for example, from different NRPS systems. In addition, the tolerance of T domain order can be tested for C domains and aminoacyl transferases by reverse-ordered tethering of native biosynthetic substrates to the native T domains and MS detection of the reaction product on the assayed upstream active site.93... 

Further recent MS applications to investigate substrate specificity were the characterization of aminoacyl-transferase CmaE in coronamic acid biosynthesis pathway106 (Figure 21(a)). Purified CmaE transferred various chemically differing aminoacyl groups between various T domains, which were detected by MALDI-TOF MS, and therefore CmaE promiscuity was identified (Figure 21(b)). 

Figure 21 Characterization of aminoacyl transferase CmaE in coronamic acid biosynthesis pathway, (a) Within the biosynthetic pathway, CmaE carries out substrate shuttling from theCmaAT domain to the CmaDT domain, (b) CmaE substrate tolerance was characterized by MALDI-TOF MS (observed and calculated mass shift of CmaD in the table). In addition, evidence of reversible aminoacyl transfer by CmaE was detected.

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