头孢菌素酰化酶晶体结构

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7-aminocephalosporanic acid (GL-7-ACA) to produce 7-aminocephalosporanic acid (7-ACA). 7-ACA is a key intermediate for the synthesis of the industrial production of most semisynthetic cephem antibiotics. 7-ACA is usually produced by chemical deacylation of cephalosporin C (CPC). However, comparison conventional chemical method, enzymatic method is simple, convenient, moderate and environment friendly. Cephalosporin acylases is a member of the N-terminal nucleophilic hydrolase, and is activated by intramolecular autoproteolytic processing. .So far, the activity of cephalosporin acylase is quite low and its Km value is in the mM range. The acylase from Pseudomonas sp. strain 130 (CA-130) is highly active on GL-7-ACA, but showed low activity for CPC. The detailed determination of the three-dimensional structure of this enzyme, the research of the relationship between the structure and functions and the site-directed mutagenesis for definite purpose will provide a new approach and a theoretical basis for improving the activity of this enzyme, so that an improved strain of cephalosporin acylase which is more stable and high-expressed is able to be obtained, the industrialization of producing cephem antibiotices with enzyme can be realized..CA-130 was crystallized in two different forms, which are both suitable for structural studies. A tetragonal crystal form diffracted to 2.4 ? and belongs to the space group P41212, with unit-cell parameters a=b=73.5?, c=382.0?. It is given that there are one ab heterodimer per asymmetric unit. A second crystal form diffracted to 2.1 ? and belongs to the space group P21, with unit-cell parameters a=130.6 ?, b=108.6 ?, c=135.5 ?, b=116.0°. It is given that there are four ab heterodimers per asymmetric unit, and furthermore every two heterodimers form a dimer. The tetragonal crystal structure of CA-130 was determined by Multiple-wavelength Anomalous Diffraction (MAD) method, and the P21 crystal structure was then determined by Molecular Replacement method. The difference in space group and unit-cell gives rise to two distinct arrangements of the protein in the respective unit cells. Comparison of the crystal packing in the two crystal forms also shows the significant change in the crystal contact.The overall structure looks like a butterfly. The active site is mostly formed by the distinctive structural motif of the N-terminal hydrolase superfamily. This structure provides detailed insight into the key residues responsible for the specificity of the CPC side chain in CA-130, and it thereby forms a basis for the design of an enzyme with an improved conversion rate of CPC to 7-ACA. This structure also provides deep insight into the structure and function of cephalosporin acylase and other N-terminal nucleophilic hydrolase..A subtle difference in the side chain orientation of Ser1b reveals a probable Ser-His-Glu triad near the active site. Based on the crystal structure, site-directed mutagenesis and subsequent characterization of mutant enzymes demonstrates that His23b is essential for autoproteolysis while Glu455b is responsible for the efficiency of the process. Although this apparent Ser-His-Glu triad is not essential for the acylase activity, His23b is also important for substrate binding and affects the catalytic efficiency..Recently, it is found that CA-130 can be affinity alkylated by 7b-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7b-3-bromopropiony aminocephalosporanic acid (BP-7-ACA) by our group. The structure determination of the protein complex with BA-7-ACA and BP-7-ACA is being carried on, which may demonstrate the activation mechanism of this enzyme more deeply...

头孢菌素酰化酶类是能够裂解头孢菌素侧链与其母核，即7－氨基头孢烷酸之间的酰氨键的饷福?－氨基头孢烷酸又是半合成β－内酰胺类抗生素的重要母核。精确测定头孢菌素酰化酶的二维结构，明确催化部位及其催化机理，并在此基础上制备一系列的突变体及复合物，测定其晶体结构，以期获得稳定而又有高催化活性及高底物专一性酶，为实现酶法合成7－氨基头孢烷酸的工业化奠定基础。.