A Novel Mitochondrial Serine O-Acetyltransferase, OpSAT1, Plays a Critical Role in Sulfur Metabolism in the Thermotolerant Methylotrophic Yeast Ogataea parapolymorpha

Ji Yoon Yeon,Su Jin Yoo,Hyun Ah Kang,Hiroshi Takagi

doi:10.1038/s41598-018-20630-8

Ji Yoon Yeon, Su Jin Yoo + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-20630-8

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Abstract

In most bacteria and plants, direct biosynthesis of cysteine from sulfide via O-acetylserine (OAS) is essential to produce sulfur amino acids from inorganic sulfur. Here, we report the functional analysis of a novel mitochondrial serine O-acetyltransferase (SAT), responsible for converting serine into OAS, in the thermotolerant methylotrophic yeast Ogataea parapolymorpha. Domain analysis of O. parapolymorpha SAT (OpSat1p) and other fungal SATs revealed that these proteins possess a mitochondrial targeting sequence (MTS) at the N-terminus and an α/β hydrolase 1 domain at the C-terminal region, which is quite different from the classical SATs of bacteria and plants. Noticeably, OpSat1p is functionally interchangeable with Escherichia coli SAT, CysE, despite that it displays much less enzymatic activity, with marginal feedback inhibition by cysteine, compared to CysE. The Opsat1Δ-null mutant showed remarkably reduced intracellular levels of cysteine and glutathione, implying OAS generation defect. The MTS of OpSat1p directs the mitochondrial targeting of a reporter protein, thus, supporting the localization of OpSat1p in the mitochondria. Intriguingly, the OpSat1p variant lacking MTS restores the OAS auxotrophy, but not the cysteine auxotrophy of the Opsat1Δ mutant strain. This is the first study on a mitochondrial SAT with critical function in sulfur assimilatory metabolism in fungal species.

Highlights

Plant, bacterial, and algal serine O-acetyltransferase (SAT) show high similarity in domain structure, with all of them carrying the N-terminal domain of serine acetyltransferase (SATase_N), which is associated with enzymatic activity, and the C-terminal hexapeptide repeat domain, which is involved in the formation of the SAT-cysteine synthase
To obtain comprehensive information on the structural features and evolutionary origin of O. parapolymorpha Sat1p (OpSat1p), we constructed the phylogenetic tree of SATs from various organisms, including yeast, filamentous fungi, protists, algae, plants, and bacteria
Most protist SATs only contain the hexapeptide repeat domain, with the exception of E. dispar SAT1. This clearly indicates that the SATs of yeasts and filamentous fungi have evolved divergently from those previously characterized from other organisms

Summary

Introduction

Bacterial, and algal SATs show high similarity in domain structure, with all of them carrying the N-terminal domain of serine acetyltransferase (SATase_N), which is associated with enzymatic activity, and the C-terminal hexapeptide repeat domain, which is involved in the formation of the SAT-cysteine synthase To investigate the in vitro activity of OpSat1p in comparison with E. coli CysE, both SATs were expressed as recombinant proteins fused with glutathione S-transferase (GST) at the N-terminus.

Results

Conclusion