Analysis of stereospecificity control mechanism in microbial lactonohydrolase.

通过对来自真菌尖孢镰刀菌（Fusarium oxysporum）和细菌原激素短杆菌（Brevibacterium protophormiae）的酶的结构-功能分析，阐明了微生物立体专一性内酯水解酶的反应和立体专一性控制机制。NH_4的氨基酸序列确定酶的2个末端和内部肽片段以制备合成的寡核苷酸作为PCR的引物。将如此扩增的000碱基基因组DNA片段用作探针以克隆该酶的基因组DNA和cDNA。内酯水解酶基因组基因由六个外显子组成，由五个短内含子分隔。在PEG 4，000作为沉淀剂的存在下，通过气相扩散程序使酶结晶。晶体属单斜晶系，空间群P2_1，晶胞参数a = 156，B = 100，c = 94.1，β = 91.7。对原激素B.的酶进行了分离和一些详细的表征。测定了该酶NH_2端和内部肽段的氨基酸序列。基于这些序列数据，制备合成的寡核苷酸作为PCR的引物，扩增基因组DNA片段，并使用该扩增片段作为探针克隆该酶的基因组DNA。

Elucidation of the reaction and stereospecificity-control mechanisms of microbial stereospecific lactonohydrolases were carried out through structure-function analyses of the enzymes from a fungal strain Fusarium oxysporum and a bacterial strain Brevibacterium protophormiae.As to the enzyme of F.oxysporum, the amino acid sequences of the NH_2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR.An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. The enzyme was crystallized by the vapor-diffusion procedure in the presence of PEG 4,000 as a precipitant. The crystals belong to the monoclinic space group P2_1 with unit-cell parameters a = 156, b = 100, c = 94.1 A, beta = 91.7。.The enzyme of B.protophormiae was isolated and characterized in some detail. The amino acid sequences of NH_2 terminus and internal peptide fragments of the enzyme were determined. On based on these sequence data, synthetic oligonucleotides were prepared as primers for the PCR.Genomic DNA fragment was amplified, and cloning of genomic DNA for the enzyme using this amplified fragment as the probe is now in progress.