Shandong Provincial Key Laboratory of Applied Mycology, College of Life Sciences, Qingdao Agricultural University, Qingdao, People's Republic of China.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
Appl Environ Microbiol. 2020 Aug 18;86(17). doi: 10.1128/AEM.01186-20.
DSM 3043 can grow on ,-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the , , , and genes are responsible for DMG degradation to sarcosine. The gene heterologously expressed in was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD to NADP The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG mutants (, , , and ) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. C nuclear magnetic resonance (C-NMR) experiments revealed that not only ectoine, glutamate, and -acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants. Although ,-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in .
DSM 3043 可以在 - 二甲基甘氨酸 (DMG) 上生长，作为唯一的 C、N 和能源，并在有氧条件下利用肌氨酸作为唯一的 N 源。然而，目前对于该菌株中 DMG 转化为肌氨酸所涉及的基因和酶知之甚少。在本研究中，基因敲除和互补测定表明， ， ， 和 基因负责 DMG 降解为肌氨酸。在 中异源表达的 基因被证明编码一种不寻常的 DMG 脱氢酶 (DMGDH)。该酶以非共价结合黄素腺嘌呤二核苷酸的 79 kDa 单体形式存在，可同时作为 DMG 和肌氨酸的底物，并表现出双重辅酶特异性，优先选择 NAD 而不是 NADP。酶活性的最佳 pH 和温度分别确定为 7.0 和 60°C。相应地确定了酶对其底物的动力学参数。在高盐条件下，DMG 的存在抑制了野生型的生长，并诱导了细胞内海藻糖和葡糖基甘油酸的产生和积累。此外，外源添加 DMG 可显著提高四种 DMG 突变体（ ， ， ，和 ）在含有肌氨酸作为唯一 N 源的 S-M63 合成培养基中于 37°C 下的生长速率。13C 核磁共振 (13C-NMR) 实验表明，不仅是章鱼胺、谷氨酸和 -乙酰-2,4-二氨基丁酸，而且还有甜菜碱 (GB)、DMG、肌氨酸、海藻糖和葡糖基甘油酸都在四种突变体的细胞内积累。尽管在微生物细胞提取物中检测到了 - 二甲基甘氨酸 (DMG) 脱氢酶 (DMGDH) 活性，但直到现在才确定编码微生物 DMGDH 的基因。此外，据我们所知，DMG 在中度嗜盐菌中的生理作用从未被研究过。在本研究中，我们鉴定了参与 DMG 降解为肌氨酸的基因，表征了一种不寻常的 DMGDH，并研究了 DMG 在 DSM 3043 及其突变体中的作用。我们的结果表明，DMGDH 中电子的分子内传递伴随着 DMG 向肌氨酸的转化，以及 DMGDH 与其他电子受体之间的分子间电子转移。此外，预测细胞中存在一种不与报道的肌氨酸 DMG 甲基转移酶同源的未知甲基转移酶，它催化 DMG 生成甜菜碱 (GB)。本研究的结果扩展了我们对 DMG 的生理作用及其在 中的代谢为肌氨酸的理解。
