The GlnD and GlnK homologues of Streptomyces coelicolor A3(2) are functionally dissimilar to their nitrogen regulatory system counterparts from enteric bacteria
The GlnD and GlnK homologues of Streptomyces coelicolor A3(2) are functionally dissimilar to their nitrogen regulatory system counterparts from enteric bacteria
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DOI：
10.1046/j.1365-2958.2002.03149.x
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发表时间：
2002-10-01
影响因子：
3.6
通讯作者：
Engels, A
中科院分区：
文献类型：
--
作者：
Hesketh, A;Fink, D;Engels, A
天蓝色链霉菌中的谷氨酰胺合成酶I（GSI）酶活性与肠道细菌中一样，在培养后由腺苷酰转移酶（GlnE）控制。虽然大肠杆菌Ntr系统的其它同源物（glnK，编码PII家族蛋白;和glnD，编码尿苷酰转移酶）在S. coelicolor基因组中，GSI活性的调节被发现是不同的。通过特异性突变体分析glnK和glnD的功能。令人惊讶的是，生物化学测定和二维PAGE分析表明，GlnE修饰GSI正常发生在所有突变株中，并且GlnK和GlnD都不是响应于氮刺激的GlnE调节所必需的。通过二维PAGE和质谱法分析GlnK在体内的翻译后调节表明，它受到可逆和不可逆的修饰，直接响应于氮的可用性。不可逆的修饰被鉴定为去除蛋白质的前三个N-末端氨基酸残基，而可逆的修饰被鉴定为保守的酪氨酸51残基的腺苷酸化，该残基已知在E.杆菌仅表达GlnD的N-末端一半的GlnD插入突变体能够使GlnK腺苷酸化，但不能响应于过量的铵进行反向去腺苷酸化反应。glnD无效突变体完全缺乏腺苷酸化GlnK的能力。这项工作提供了通过腺苷酸化修饰的PII蛋白的第一个例子，并证明该反应是由GlnD的同源物进行的，先前仅描述为尿苷酰转移酶。
Glutamine synthetase I (GSI) enzyme activity in Streptomyces coelicolor is controlled post-translationally by the adenylyltransferase (GlnE) as in enteric bacteria. Although other homologues of the Escherichia coli Ntr system (glnK, coding for a PII family protein; and glnD, coding for an uridylyltransferase) are found in the S. coelicolor genome, the regulation of the GSI activity was found to be different. The functions of glnK and glnD were analysed by specific mutants. Surprisingly, biochemical assay and two-dimensional PAGE analysis showed that modification of GSI by GlnE occurs normally in all mutant strains, and neither GlnK nor GlnD are required for the regulation of GlnE in response to nitrogen stimuli. Analysis of the post-translational regulation of GlnK in vivo by two-dimensional PAGE and mass spectrometry indicated that it is subject to both a reversible and a non-reversible modification in a direct response to nitrogen availability. The irreversible modification was identified as removal of the first three N-terminal amino acid residues of the protein, and the reversible modification as adenylylation of the conserved tyrosine 51 residue that is known to be uridylylated in E. coli. The glnD insertion mutant expressing only the N-terminal half of GlnD was capable of adenylylating GlnK, but was unable to perform the reverse deadenylylation reaction in response to excess ammonium. The glnD null mutant completely lacked the ability to adenylylate GlnK. This work provides the first example of a PII protein that is modified by adenylylation, and demonstrates that this reaction is performed by a homologue of GlnD, previously described only as a uridylyltransferase enzyme.