A novel arginine metabolic pathway regulated by the yeast
N-acetyltransferase Mpr1
Ryo NASUNO
Graduate School of Biological Sciences, Nara Institute of Scienceand Technology

Report: The yeast N-acetyltransferase Mpr1 enhances arginine production via
N-acetyl-glutamic acid synthesis by inducing the transcription of ornithine acetyltransferase
Arg7.
Research aims
Recent studies have focused on the physiological functions of free amino acids inaddition to the function of complete proteins. Previously, we demonstrated that the yeast
N-acetyltransferase Mpr1 confers oxidative stress tolerance to yeast via arginine
biosynthesis1 and determined its crystal structure. We also succeeded in enhancing
Mpr1-dependent arginine production by thermostabilization using a structure-based rational
design2. However, the mechanism underlying Mpr1-dependent arginine synthesis remains
unclear. This study aimed to clarify the molecular mechanism of the Mpr1-dependentarginine biosynthesis pathway.
Methods
The yeast Saccharomyces cerevisiae L5685 strain, derived from Σ1278b strain andharboring the MPR1 gene in its genome, was used as a wild-type strain in this study. Thegene disruption was performed via homologous recombination using an antibiotic resistancegene. Arg5 and Arg6 localize and function in the mitochondria and are encoded in theARG5,6 single gene. Therefore, the Arg6-deficient strain was constructed by deletion ofARG5,6 gene followed by the introduction of the plasmid expressing the ARG5,6 geneencoding Arg6 with an amino acid substation Asp251Glu in its catalytic center. MPR1
overexpression was performed via the multi-copy type plasmid harboring the MPR1 gene
under the control of a GPD promoter. The yeast cell was cultured in a complete synthetic
medium at 30 °C by omitting designated amino acids and/or nucleic acid bases, as required.
RT-PCR was performed to analyze the transcription level of ARG7. The recombinant
purified Mpr1 from E. coli was used for its enzymatic reaction in vitro. Novel substrates ofMpr1 were screened via colorimetric assay using Ellman’s reagent to detect its reaction
product CoA. The reaction product N-acetyl-proline was identified via LC-MS analysis.
ResultsThe S. cerevisiae ∆arg2 strain
showed remarkable growth defect under Argdepleted conditions (Arg auxotrophy). Our
previous study showed that Mpr1 is involvedin one of the steps between Arg2 and Arg8 in
the arginine biosynthesis pathway (Figure).
The growth of ∆arg2 and ∆arg5,6 strains wasevaluated in order to examine which specificstep involves Mpr1. MPR1 overexpression
compensated for the Arg auxotrophy of ∆arg2
but not of the ∆arg5,6 strain. Subsequent
analysis showed that MPR1 overexpression
did not compensate for Arg auxotrophy of thestrain expressing the unfunctional Arg6
mutant which has the amino acid substitution
at its catalytic center. In contrast, the Arg
auxotrophy of the ∆arg2 strain was reversedby supplementation of N-acetyl-glutamate(NAG) to the medium. Interestingly, NAG isthe reaction product of the Arg-catalyzed
enzymatic reaction. These results suggested
that Mpr1 accelerates Arg biosynthesis via
NAG production.
Ornithine acetyltransferase (OAT)
Arg7, which catalyzes the deacetylation
reaction of N-acetyl-ornithine, had beensuggested to exert NAGS activity like Arg23. The overexpression of ARG7 compensated forArg auxotrophy of the ∆arg2 strain, indicating that Arg7 possesses NAGS activity. RT-PCR
analysis indicated that MPR1 overexpression induced the transcription of ARG7. On thebasis of the results of this study, we conclude that Mpr1 increases NAG production via
Arg7’s NAGS activity, which induces the transcription of ARG7, leading to the enhancementof Arg biosynthesis.

Conclusion
In this study, we used a growth test to evaluate Arg auxotrophy using arginine
biosynthesis gene disruptants, mutants, and RT-PCR analysis. We found that Mpr1 increases
NAG production through an increase in NAGS activity in Arg7, which occurs via
transcriptional induction of ARG7, resulting in the enhancement of Arg biosynthesis. Our
previous study suggested that Mpr1 exists in both the cytosol and mitochondria; no reports
have suggested that Mpr1 translocates to the nucleus. Screening for novel substrates of Mpr1
performed in this study found that proline is converted to N-acetyl-proline by Mpr1 underbasic conditions. As of now, there are no studies on the biological significance ofN-acetyl-proline. Thus, its physiological functions are unclear. However, it is possible thatN-acetyl-proline is involved in Mpr1-dependent ARG7 induction. Indeed, since someN-acetyltransferases acetylate both small molecules and proteins as their substrates4, Mpr1
might induce ARG7 similarly, by acetylating certain proteins.
In future studies, Arg7 protein levels will be evaluated in order to test theconclusion made here. Localization analysis of Mpr1 and/or chromatin immunoprecipitation
analysis is required to examine whether Mpr1 is directly involved in ARG7 induction.
Physiological analyses of N-acetyl-proline and evaluation of N-acetylation of the proteins
involved in ARG7 induction by Mpr1 will be performed to clarify the molecular mechanismof Mpr1-dependent ARG7 transcriptional upregulation.
References
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mechanism mediated by the yeast N-acetyltransferase Mpr1: oxidative stress-induced
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