The phospholipase pl-S.t gene of Sphingobacterium thalpophilum 2015 was cloned and the gene sequence was submitted to NCBI with Accession Number KX674735.1. The phylogenetic analysis showed that this PL-S.t was clustered to phospholipase D (PLD). As far as we know, the PL-S.t with a molecular mass of 22.5 kDa is the lowest of the currently purified bacterial PLDs, which belongs to a non-HKD PLD enzyme. This PL-S.t was resistant to a wide range of alkali pHs (7.5–9.0) after 1 h incubation, retaining more than 90% of its maximum activity. The PL-S.t activity can be enhanced by Ni2+, Co2+ and Mn2+. This PL-S.t has only one cysteine residue and fewer negatively-charged amino acids (AAs). The hydrogen bonds network was found around the cystein108, which may be beneficial to the stability and activity of PL-S.t in Ni2+ solution. This study has laid the foundation for further research on the molecular mechanism of the catalytic characteristics of low molecular weight alkalic PLD from S. thalpophilum 2015.
The yeast Yarrowia lipolytica assimilates hydrophobic compounds, such as n-alkanes and fatty acids, as sole carbon and energy sources. It has been shown that the acyl-CoA synthetase (ACS) genes, FAT1 and FAA1, are involved in the activation of fatty acids produced during the metabolism of n-alkanes, but the ACS genes that are involved in the metabolism of fatty acids from the culture medium remains to be identified. In this paper, we have identified the ACS genes involved in the utilization of exogenous fatty acids. RNA-seq analysis and qRT-PCR revealed that the transcript levels of the peroxisomal ACS-like protein-encoding genes AAL4 and AAL7 were increased in the presence of oleic acid. The single deletion mutant of AAL4 or AAL7 and double deletion mutant of AAL4 and AAL7 did not show any defects in the growth on the medium containing glucose, glycerol, n-alkanes, or fatty acids. In contrast, the mutant with deletion of seven genes, FAA1, FAT1-FAT4, AAL4, and AAL7, showed severe growth defects on the medium containing dodecanoic acid or oleic acid. These results suggest that Aal4p and Aal7p play important roles in the metabolism of exogenous fatty acids in collaboration with Faa1p and Fat1p-Fat4p.
Glutaredoxins (Grxs) with Cys-Pro-Phe (Tyr)-Cys motif and a thioredoxin fold structure play an important role in the anti-oxidant system of bacteria by catalyzing a variety of thiol-disulfide exchange reactions with a 2-Cys mechanism or a 1-Cys mechanism. However, the catalytic and physiological mechanism of Corynebacterium glutamicum Mycoredoxin 1 (Mrx1) that shares a high amino acid sequence similarity to Grxs has not been fully elucidated. Here, we report that Mrx1 has a protective function against various adverse conditions, and the decrease of cell viability to various stress conditions by deletion of the Mrx1 in C. glutamicum was confirmed in the mrx1 mutant. The physiological roles of Mrx1 in defence to oxidative stress were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. As well as reducing mycothiol (MSH) mixed disulfide bonds via a 1-Cys mechanism, C. glutamicum Mrx1 catalytically reduced the disulfides in the Ib RNR, insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by exclusively linking the MSH/Mtr (mycothiol disulfide reductase)/NADPH electron pathway via a 2-Cys mechanism. Thus, we present the first evidence that the Mrx1 is able to protect against the damaging effects of various exogenous stresses by acting as a disulfide oxidoreductase, thereby giving a new insight in how C. glutamicum survives oxidative stressful conditions.
Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.
O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.
The firefly luciferase (Luc) reporter assay is a powerful tool used to analyze promoter activities in living cells. In this report, we established a firefly Luc reporter assay system in the unicellular model red alga Cyanidioschyzon merolae. A nitrite reductase (NIR) promoter-Luc fusion gene was integrated into the URA5.3 genomic region to construct the C. merolae NIR-Luc strain. Luc activities in the NIR-Luc strain were increased, correlating with the accumulation of endogenous NIR transcripts in response to nitrogen depletion. Luc activity was also significantly increased by the overexpression of the MYB1 gene, which encodes a transcription factor responsible for NIR promoter activation. Thus, our results demonstrate the utility of the Luc reporter system in C. merolae.