L-lactate 2-monooxygenase from Mycobacterium smegmatis. Cloning, nucleotide sequence, and primary structure homology within an enzyme family.
L-lactate 2-monooxygenase from Mycobacterium smegmatis. Cloning, nucleotide sequence, and primary structure homology within an enzyme family.
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DOI：
10.1016/s0021-9258(19)39195-1
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发表时间：
1990-04
期刊：
影响因子：
--
通讯作者：
D. Giegel;C. Williams;V. Massey
中科院分区：
文献类型：
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作者：
D. Giegel;C. Williams;V. Massey
L-乳酸2-单加氧酶催化L-乳酸氧化为乙酸和二氧化碳。催化机制已被广泛研究，但很少有人知道哪些氨基酸残基可能在催化中发挥作用。作为实现这一目标的第一步，来自耻垢分枝杆菌的这种蛋白质的基因已被克隆和测序。利用L-乳酸2-单加氧酶的肽序列数据构建三组完全冗余的十四聚体寡核苷酸探针，将其与限制性消化的M.包皮垢DNA。一个大约3-磷酸酶对Pst I片段与两个探针杂交。该区域随后被分离并克隆到大肠杆菌中。从这个大小分级的基因库中，通过与两个寡核苷酸探针的菌落杂交分离3.1-内切酶对基因组DNA片段。如整个插入片段的DNA测序所示，L-乳酸2-单加氧酶的完整基因包含在该片段上。该DNA序列编码一个成熟蛋白，其长度为393个氨基酸，亚基分子量为43，072（包括FMN）。该蛋白质序列显示出与两种机制相关蛋白质，酵母黄细胞色素b2（Lederer，F.，Cortial，S.，Becam，A. - M.，Haumont，P.是的，和Perez，L.（1985）Eur. 152，419 - 428; Guiard，B.（1985）EMBO J.4，3265 - 3272）和菠菜乙醇酸氧化酶（Volkita，M.，和萨默维尔，C. R.（1987）J.Biol.Chem.262，15825 - 15828; Cederlund，E.，Lindqvist，Y.，Soderlund，G.，Bränden角一、Jornvall，H.（1988）Eur. 173，523 - 530）。对于从乙醇酸氧化酶的晶体结构中提出的参与催化的每个残基（Lindqvist，Y.，和Brändén，C. - I.（1989）J.Biol.Chem.264，3624 - 3628），在乳酸氧化酶的同源位置发现了相同的残基。此外，这些残基中的大多数发生在其序列在乳酸氧化酶、黄细胞色素b2和乙醇酸氧化酶之间高度保守的区域中。
L-Lactate 2-monooxygenase catalyzes the oxidation of L-lactate to acetate and carbon dioxide. The catalytic mechanism has been extensively investigated but very little is known about which amino acid residues may play a role in catalysis. As a first step toward this goal, the gene for this protein from Mycobacterium smegmatis has been cloned and sequenced. Peptide sequencing data for L-lactate 2-monooxygenase was used to construct three sets of fully redundant tetradecamer oligonucleotide probes, which were hybridized to restriction-digested M. smegmatis DNA. An approximately 3-kilobase pair PstI fragment hybridized with two of the probes. This region was subsequently isolated and cloned into Escherichia coli. From this size-fractionated gene bank, a 3.1-kilobase pair genomic DNA fragment was isolated by colony hybridization to two of the oligonucleotide probes. The complete gene for L-lactate 2-monooxygenase was contained on this fragment as shown by DNA sequencing of the whole insert. The DNA sequence codes for a mature protein that is 393 amino acids in length with a subunit molecular weight of 43,072 (including the FMN). The protein sequence shows impressive homology with the primary structures of two mechanistically related proteins, yeast flavocytochrome b2 (Lederer, F., Cortial, S., Becam, A.-M., Haumont, P.-Y., and Perez, L. (1985) Eur. J. Biochem. 152, 419-428; Guiard, B. (1985) EMBO J. 4, 3265-3272) and spinach glycolate oxidase (Volkita, M., and Somerville, C. R. (1987) J. Biol. Chem. 262, 15825-15828; Cederlund, E., Lindqvist, Y., Soderlund, G., Brändén, C.-I., and Jornvall, H. (1988) Eur. J. Biochem. 173, 523-530). For each residue proposed from the crystal structure of glycolate oxidase to be involved in catalysis (Lindqvist, Y., and Brändén, C.-I. (1989) J. Biol. Chem. 264, 3624-3628), an identical residue was found in a homologous position in lactate oxidase. Furthermore, most of these residues occur in regions whose sequences are highly conserved between lactate oxidase, flavocytochrome b2, and glycolate oxidase.