Bompard-Gilles C, Remaut H, Villeret V, Prangé T, Fanuel L, Delmarcelle M, Joris B, Frère J, Van Beeumen J
Laboratorium voor Eiwitbiochemie en Eiwitengineering, Rijksuniversiteit-Gent, K.L. Ledeganckstraat, 35, B-9000, Gent, Belgium.
Structure. 2000 Sep 15;8(9):971-80. doi: 10.1016/s0969-2126(00)00188-x.
beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases.
The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme.
Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique.
β-内酰胺类化合物是使用最广泛的抗生素。它们能使参与细胞壁生物合成的细菌DD-转肽酶（也称为青霉素结合蛋白，PBPs）失活。细菌对β-内酰胺类化合物最常见的耐药机制是合成水解β-内酰胺环的β-内酰胺酶。据信这些酶是从细胞壁DD-肽酶进化而来的。由于耐药细菌数量不断增加，了解β-内酰胺靶点的生化和机制特征至关重要。DAP是嗜水气单胞菌产生的一种D-氨基肽酶。它受到多种β-内酰胺类化合物的抑制，与R61 DD-羧肽酶和C类β-内酰胺酶具有约25%的序列同一性。
采用多同晶置换（MIR）方法，已将DAP的晶体结构解析到1.9 Å分辨率。该酶折叠成A、B和C三个结构域。包含保守催化残基的结构域A具有丝氨酸β-内酰胺酶的经典折叠结构，而结构域B和C均为反平行八链β桶。结构域C的一个环突出到酶的底物结合位点中。
将DAP的生化特性和结构与PBPs及丝氨酸β-内酰胺酶进行比较表明，尽管该酶的催化位点与β-内酰胺酶非常相似，但其底物和抑制剂特异性取决于结构域C的残基。DAP是青霉素识别蛋白（PRPs）家族的新成员，目前其酶促特异性明显独特。
