RESUMEN
The N-acetylmuramic acid α-1-phosphate (MurNAc-α1-P) uridylyltransferase MurU catalyzes the synthesis of uridine diphosphate (UDP)-MurNAc, a crucial precursor of the bacterial peptidoglycan cell wall. MurU is part of a recently identified cell wall recycling pathway in Gram-negative bacteria that bypasses the general de novo biosynthesis of UDP-MurNAc and contributes to high intrinsic resistance to the antibiotic fosfomycin, which targets UDP-MurNAc de novo biosynthesis. To provide insights into substrate binding and specificity, we solved crystal structures of MurU of Pseudomonas putida in native and ligand-bound states at high resolution. With the help of these structures, critical enzyme-substrate interactions were identified that enable tight binding of MurNAc-α1-P to the active site of MurU. The MurU structures define a "minimal domain" required for general nucleotidyltransferase activity. They furthermore provide a structural basis for the chemical design of inhibitors of MurU that could serve as novel drugs in combination therapy against multidrug-resistant Gram-negative pathogens.
Asunto(s)
Nucleotidiltransferasas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/patogenicidad , Magnesio/química , Modelos Moleculares , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Especificidad por Sustrato , Uridina Difosfato Ácido N-Acetilmurámico/biosíntesisRESUMEN
The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host-pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding.