McLean Jeremy E, Hamaguchi Nobuko, Belenky Peter, Mortimer Sarah E, Stanton Martin, Hedstrom Lizbeth
Program in Biophysics and Structural Biology, Brandeis University, MS 009, 415 South St., Waltham, MA 02454, USA.
Biochem J. 2004 Apr 15;379(Pt 2):243-51. doi: 10.1042/BJ20031585.
Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.
肌苷5'-单磷酸脱氢酶（IMPDH）是鸟嘌呤核苷酸从头生物合成中的限速酶。除催化结构域外，IMPDH还包含一个由两个胱硫醚β-合酶结构域组成的功能未知的亚结构域。我们使用三种不同检测方法得到的结果表明，胎儿三毛滴虫、大肠杆菌以及两种人类同工型的IMPDH通过该亚结构域以纳摩尔亲和力结合单链核酸。每个IMPDH四聚体大约结合100个核苷酸。亚结构域的缺失使亲和力降低10倍，并使结合位点大小降至60个核苷酸，而用Glu取代亚结构域中保守的Arg/Lys残基会使亲和力降低20倍。正如对核酸结合蛋白所预期的那样，在人类细胞核中发现了IMPDH。最后，免疫沉淀实验表明，IMPDH在体内能结合RNA和DNA。这些实验表明，IMPDH在由该亚结构域介导的复制、转录或翻译过程中具有此前未被认识到的作用。
