Regulation of d-Aspartate Oxidase Gene Expression by Pyruvate Metabolism in the Yeast Cryptococcus humicola.

Daiki Imanishi,Shouji Takahashi,Sota Zaitsu

doi:10.3390/microorganisms9122444

Abstract

d-Aspartate oxidase (DDO) is a peroxisomal flavoenzyme that catalyzes the oxidative deamination of acidic d-amino acids. In the yeast Cryptococcus humicola strain UJ1, the enzyme ChDDO is essential for d-Asp utilization and is expressed only in the presence of d-Asp. Pyruvate carboxylase (Pyc) catalyzes the conversion of pyruvate to oxaloacetate and is involved in the import and activation of certain peroxisomal flavoenzymes in yeasts. In this study, we analyzed the role of Pyc in the expression of ChDDO gene in C. humicola strain UJ1. PYC gene disruption (∆Chpyc1) in strain UJ1 resulted in growth retardation on glucose and NH4Cl medium. The growth was restored by supplying oxaloacetate from l-Asp or α-ketoglutarate by a transaminase. On the other hand, the supply of oxaloacetate from d-Asp by ChDDO was not able to prevent growth retardation because of a significant decrease in ChDDO gene expression at the transcriptional level. The addition of pyruvate significantly decreased ChDDO gene transcription in the ∆Chpyc1 strain but increased the same in the wild-type strain, even though the intracellular pyruvate content was similar in both strains. These results suggest that ChDDO gene expression might be regulated by pyruvate metabolism, as well as by the presence of d-Asp.

Highlights

To identify a Pyruvate carboxylase (Pyc) homolog in C. humicola strain UJ1, we searched for a homolog in the draft genome sequence of strain UJ1 using the amino acid sequence of Pyc1 (PpPyc1, Uniprot: P78992) of the yeast P. pastoris
Three conserved functional domains of Pyc namely N-terminal biotin carboxylation (BC) domain, central transcarboxylation (TC) domain, and C-terminal biotin carboxyl carrier (BCC) domain were found in ChPyc1 (Figure 1) [38], suggesting that ChPyc1 might function as Pyc in strain UJ1
The ∆Chpyc1 strain showed growth retardation on a minimal medium containing glucose and NH4 Cl as the sole carbon and nitrogen sources, respectively, whereas the growth of the ∆Chpyc1 strain was restored by the addition of L-Asp or α-ketoglutarate (Figure 4). These results indicate that ChPyc1 can function as an anaplerotic enzyme of the TCA cycle and that, instead of ChPyc1, the Asp transaminase reaction can complement the function of Pyc by supplying oxaloacetate to the TCA cycle

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Oxidase (DDO, EC 1.4.3.1) catalyzes the oxidative deamination of acidic acids to yield corresponding α-keto acids and ammonia. The same reaction for neutral and basic D-amino acids is catalyzed by D -amino acid oxidase (DAO, EC 1.4.3.3). Both enzymes are localized in peroxisomes and require flavin adenine dinucleotide (FAD)

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