Ishigaki Yuji, Akanuma Genki, Yoshida Minoru, Horinouchi Sueharu, Kosono Saori, Ohnishi Yasuo
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan.
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Proteomics. 2017 Feb 23;155:63-72. doi: 10.1016/j.jprot.2016.12.006. Epub 2016 Dec 26.
Protein acetylation, the reversible addition of an acetyl group to lysine residues, is a protein post-translational modification ubiquitous in living cells. Although the involvement of protein acetylation in the regulation of primary metabolism has been revealed, the function of protein acetylation is largely unknown in secondary metabolism. Here, we characterized protein acetylation in Streptomyces griseus, a streptomycin producer. Protein acetylation was induced in the stationary and sporulation phases in liquid and solid cultures, respectively, in S. griseus. By comprehensive acetylome analysis, we identified 134 acetylated proteins with 162 specific acetylated sites. Acetylation was found in proteins related to primary metabolism and translation, as in other bacteria. However, StrM, a deoxysugar epimerase involved in streptomycin biosynthesis, was identified as a highly acetylated protein by 2-DE-based proteomic analysis. The Lys70 residue, which is critical for the enzymatic activity of StrM, was the major acetylation site. Thus, acetylation of Lys70 was presumed to abolish enzymatic activity of StrM. In accordance with this notion, an S. griseus mutant producing the acetylation-mimic K70Q StrM hardly produced streptomycin, though the K70Q mutation apparently decreased the stability of StrM. A putative lysine acetyltransferase (KAT) SGR1683 in S. griseus, as well as the Escherichia coli KAT YfiQ, acetylated Lys70 of StrM in vitro. Furthermore, absolute quantification analysis estimated that 13% of StrM molecules were acetylated in mycelium grown in solid culture for 3days. These results indicate that StrM acetylation is of biological significance. We propose that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus.
Protein acetylation has been extensively studied not only in eukaryotes, but also in prokaryotes. The acetylome has been analyzed in more than 14 bacterial species. Here, by comprehensive acetylome analysis, we showed that acetylation was found in proteins related to primary metabolism and translation in Streptomyces griseus, similarly to other bacteria. However, five proteins involved in secondary metabolism were also identified as acetylated proteins; these proteins are enzymes in the biosynthesis of streptomycin (StrB1 and StrS), grixazone (GriF), a nonribosomal peptide (NRPS1-2), and a siderophore (AlcC). Additionally, StrM in streptomycin biosynthesis was identified as a highly acetylated protein by 2-DE-based proteomic analysis; approximately 13% of StrM molecules were acetylated. The acetylation occurs at Lys70 to abolish the enzymatic activity of StrM, suggesting that StrM acetylation functions as a limiter of streptomycin biosynthesis in S. griseus. This is the first detailed analysis of protein acetylation of an enzyme involved in secondary metabolism.
蛋白质乙酰化是指在赖氨酸残基上可逆地添加乙酰基，是一种在活细胞中普遍存在的蛋白质翻译后修饰。尽管蛋白质乙酰化在初级代谢调控中的作用已被揭示，但在次级代谢中其功能仍 largely 未知。在此，我们对链霉素产生菌灰色链霉菌中的蛋白质乙酰化进行了表征。在灰色链霉菌中，蛋白质乙酰化分别在液体和固体培养的稳定期和孢子形成期被诱导。通过全面的乙酰化蛋白质组分析，我们鉴定出 134 个乙酰化蛋白质，具有 162 个特定的乙酰化位点。与其他细菌一样，在与初级代谢和翻译相关的蛋白质中发现了乙酰化。然而，通过基于二维电泳的蛋白质组分析，参与链霉素生物合成的脱氧糖差向异构酶 StrM 被鉴定为高度乙酰化的蛋白质。对 StrM 酶活性至关重要的 Lys70 残基是主要的乙酰化位点。因此，推测 Lys70 的乙酰化会消除 StrM 的酶活性。根据这一观点，产生乙酰化模拟物 K70Q StrM 的灰色链霉菌突变体几乎不产生链霉素，尽管 K70Q 突变明显降低了 StrM 的稳定性。灰色链霉菌中一个假定的赖氨酸乙酰转移酶（KAT）SGR1683 以及大肠杆菌 KAT YfiQ 在体外使 StrM 的 Lys70 乙酰化。此外，绝对定量分析估计，在固体培养 3 天的菌丝体中，13%的 StrM 分子被乙酰化。这些结果表明 StrM 乙酰化具有生物学意义。我们提出 StrM 乙酰化在灰色链霉菌中作为链霉素生物合成的限制因素发挥作用。
蛋白质乙酰化不仅在真核生物中，而且在原核生物中都得到了广泛研究。已经对超过 14 种细菌的乙酰化蛋白质组进行了分析。在此，通过全面的乙酰化蛋白质组分析，我们表明，与其他细菌类似，在灰色链霉菌中与初级代谢和翻译相关的蛋白质中发现了乙酰化。然而，还鉴定出五种参与次级代谢的蛋白质为乙酰化蛋白质；这些蛋白质是链霉素生物合成中的酶（StrB1 和 StrS）、格氏菌素（GriF）、一种非核糖体肽（NRPS1 - 2）和一种铁载体（AlcC）。此外，通过基于二维电泳的蛋白质组分析，链霉素生物合成中的 StrM 被鉴定为高度乙酰化的蛋白质；约 13%的 StrM 分子被乙酰化。乙酰化发生在 Lys70 以消除 StrM 的酶活性，这表明 StrM 乙酰化在灰色链霉菌中作为链霉素生物合成的限制因素发挥作用。这是对参与次级代谢的一种酶的蛋白质乙酰化的首次详细分析。
