Crystal structure of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB) in complex with the inhibitor O-isobutenyl oxalylhydroxamate.

3-isopropylmalate dehydrogenases (LeuB) belong to the leucine biosynthetic pathway and catalyze the irreversible oxidative decarboxylation of 3IPM to 2-ketoisocaproate that is finally converted into leucine by a branched-chain aminotransferase. Since leucine is an essential amino acid for humans, and it is also vital for the growth of many pathogenic bacteria, the enzymes belonging to this pathway can be considered as potential target sites for designing of a new class of antibacterial agents. We have determined the crystal structure of the Haemophilus influenzae LeuB in complex with the cofactor NAD+ and the inhibitor O-IbOHA, at 2.1 Å resolution; moreover, we have investigated the inhibitor mechanism of action by analyzing the enzyme kinetics. The structure of H. influenzae LeuB in complex with the intermediate analog inhibitor displays a fully closed conformation, resembling the previously observed, closed form of the equivalent enzyme of Thiobacillus ferrooxidans in complex with the 3IPM substrate. O-IbOHA was found to bind the active site by adopting the same conformation of 3IPM, and to induce an unreported repositioning of the side chain of the amino acids that participate in the coordination of the ligand. Indeed, the experimentally observed binding mode of O-IbOHA to the H. influenzae LeuB enzyme, reveals aspects of novelty compared to the computational binding prediction performed on M. tuberculosis LeuB. Overall, our data provide new insights for the structure-based rational design of a new class of antibiotics targeting the biosynthesis of leucine in pathogenic bacteria.

X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: a multidisciplinary characterization of catechol 1,2 dioxygenase.

Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

Expression, purification and characterisation of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB).

3-isopropylmalate dehydrogenase (3IPMDH) is the third enzyme in leucine biosynthesis and a promising target for the development of broad-spectrum antibacterial agents. We report here the expression, purification and biochemical characterisation of Haemophilus influenzae 3-isopropylmalate dehydrogenase. The observed enzyme inhibition by the reaction product NADH could represent a regulatory mechanism for 3IPMDH.