Adediran S A, Zhang Zhen, Nukaga Michiyoshi, Palzkill Timothy, Pratt R F
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Biochemistry. 2005 May 24;44(20):7543-52. doi: 10.1021/bi050136f.
The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.
β-内酰胺类抗生素通过抑制D-丙氨酰-D-丙氨酸转肽酶（DD-肽酶）发挥作用，该酶催化细菌细胞壁合成的最后一步。细菌通过多种机制对β-内酰胺类产生耐药性，其中较为重要的一种机制是产生β-内酰胺酶，即催化这些抗生素水解的酶。丝氨酸β-内酰胺酶是DD-肽酶的进化后代，保留了其大部分结构，尤其是在活性位点。在功能上，β-内酰胺酶与DD-肽酶的不同之处在于，它能够催化源自β-内酰胺的酰基酶中间体的水解，并且无法有效催化D-丙氨酰-D-丙氨酸末端肽的酰基转移反应。阴沟肠杆菌P99的C类β-内酰胺酶在结构上与链霉菌R61的DD-肽酶非常相似。先前的研究表明，β-内酰胺酶的进化，大概是从类似于R61酶的祖先DD-肽酶进化而来，包括结构变化，导致DD-肽酶底物倒数第二个D-丙氨酸残基的D-甲基取代基被排斥。这似乎是通过β-内酰胺酶中Tyr 221侧链的合适位置实现的。我们在本文中表明，将该残基突变为Gly 221会产生一种酶，与野生型β-内酰胺酶相比，它更容易水解和氨解无环D-丙氨酰底物而不是甘氨酰类似物；因此，该突变体是一种更有效的DD-肽酶。分子模拟表明，D-丙氨酰甲基紧密地契合到最初由Tyr 221侧链占据的空间中，这样做使得结合的底物呈现出与R61 DD-肽酶上相似的构象，R61 DD-肽酶有一个用于该取代基的疏水口袋。P99β-内酰胺酶的另一个突变体，即超广谱GC1酶，由于一个延伸的ω环，也有可供D-丙氨酰甲基占据的空间。然而，在这种情况下，未观察到对D-丙氨酰底物相对于甘氨酰底物的活性增强。因此，倒数第二个D-丙氨酰甲基的容纳对于有效的DD-肽酶活性是必要的，但不是充分的。
