Qi H Y, Sankaran K, Gan K, Wu H C
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Bacteriol. 1995 Dec;177(23):6820-4. doi: 10.1128/jb.177.23.6820-6824.1995.
The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.
通过比较系统发育关系较远的细菌物种中该酶的一级结构、分析突变酶的序列以及对大肠杆菌酶进行特定化学修饰，对细菌前脂蛋白二酰甘油转移酶（LGT）的结构 - 功能关系进行了研究。通过对缺乏LGT活性的大肠杆菌温度敏感型lgt突变体（菌株SK634）进行互补，分离出了含有革兰氏阳性菌金黄色葡萄球菌LGT基因（lgt）的克隆。对前脂蛋白二酰甘油修饰活性的体内和体外测定表明，互补克隆恢复了突变菌株中的前脂蛋白修饰活性。插入DNA的序列测定揭示了一个837 bp的开放阅读框，编码一个279个氨基酸的蛋白质，计算分子量为31.6 kDa。金黄色葡萄球菌LGT与大肠杆菌、鼠伤寒沙门氏菌和流感嗜血杆菌的LGT具有24%的同一性和47%的相似性。金黄色葡萄球菌LGT虽然比大肠杆菌酶短12个氨基酸，但其亲水性图谱和预测的pI（10.4）与大肠杆菌酶相似。大肠杆菌、鼠伤寒沙门氏菌、流感嗜血杆菌和金黄色葡萄球菌LGT蛋白之间的多序列比对揭示了整个分子中高度保守的氨基酸序列区域。通过PCR克隆并测序了来自大肠杆菌SK634、SK635和SK636的三个独立的lgt突变等位基因以及来自鼠伤寒沙门氏菌SE5221的一个lgt等位基因，它们在非允许温度下LGT活性均有缺陷。发现突变等位基因包含单个碱基改变，导致一个保守氨基酸被取代。这四种微生物的LGT中最长的一组无间隙相同氨基酸是H - 103 - GGLIG - 108。在大肠杆菌lgt突变体SK634中，该区域的甘氨酸 - 104突变为丝氨酸，突变生物体在生长上对温度敏感，并且在体外表现出低LGT活性。焦碳酸二乙酯以18.6 M⁻¹S⁻¹的二级速率常数使大肠杆菌LGT失活，在pH 7时，LGT活性的失活可被羟胺逆转。失活动力学与对LGT活性至关重要的单个残基（组氨酸或酪氨酸）的修饰一致。
