Identification of an Acidic Amino Acid Permease Involved in d-Aspartate Uptake in the Yeast Cryptococcus humicola.

Daiki Imanishi,Shouji Takahashi,Yoshio Kera

doi:10.3390/microorganisms9010192

Abstract

d-aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic d-amino acids, and its production is induced by d-Asp in several eukaryotes. The yeast Cryptococcus humicola strain UJ1 produces large amounts of DDO (ChDDO) only in the presence of d-Asp. In this study, we analyzed the relationship between d-Asp uptake by an amino acid permease (Aap) and the inducible expression of ChDDO. We identified two acidic Aap homologs, named “ChAap4 and ChAap5,” in the yeast genome sequence. ChAAP4 deletion resulted in partial growth defects on d-Asp as well as l-Asp, l-Glu, and l-Phe at pH 7, whereas ChAAP5 deletion caused partial growth defects on l-Phe and l-Lys, suggesting that ChAap4 might participate in d-Asp uptake as an acidic Aap. Interestingly, the growth of the Chaap4 strain on d- or l-Asp was completely abolished at pH 10, suggesting that ChAap4 is the only Aap responsible for d- and l-Asp uptake under high alkaline conditions. In addition, ChAAP4 deletion significantly decreased the induction of DDO activity and ChDDO transcription in the presence of d-Asp. This study revealed that d-Asp uptake by ChAap4 might be involved in the induction of ChDDO expression by d-Asp.

Highlights

Microorganisms 2021, 9, 192. https://D -aspartate oxidase (DDO or DASPO, EC 1.4.3.1) is a peroxisomal enzyme that catalyzes the oxidative deamination of acidic D -amino acids to produce their corresponding α-keto acids and ammonia
Our results revealed that ChAap4 might participate in D -Asp uptake into cells and, indirectly the inducible expression of ChDDO, and the protein is essential for growth on D -Asp and L -Asp at a high alkaline pH, illustrating the involvement of D -Asp uptake by acidic amino acid permease (Aap) in the inducible expression of ChDDO in the presence of D-Asp
The GXG and (F/Y)(S/A/T)(F/Y)XGXE motifs were found in TM1 and TM6, respectively (Figure 3), and these motifs are highly conserved in Aaps and involved in the interactions with the αcarboxy and α-amino groups of amino acid substrates, respectively [17,35]. These findings suggested that ChAap4 and ChAap5 might function as acidic Aaps in strain UJ1

Summary

Introduction

Microorganisms 2021, 9, 192. https://D -aspartate oxidase (DDO or DASPO, EC 1.4.3.1) is a peroxisomal enzyme that catalyzes the oxidative deamination of acidic D -amino acids to produce their corresponding α-keto acids and ammonia. DDO has been found in various eukaryotic organisms, ranging from fungi to mammals, but it is not present in prokaryotic organisms [1,2,3,4,5]. DDO genes have been cloned from various eukaryotic organisms including human and mouse, and their enzymatic properties have been extensively studied [6]. C. humicola strain UJ1 (ChDDO) is the first DDO cloned from a microorganism and has high substrate specificity and high activity for D -Asp [5,7]. DDO participates in the assimilation and detoxification of acidic D -amino acids [8]. It might be important to reveal the environmental and protein factors that can affect the expression of DDO gene

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