Studies on Structures and Reaction Mechanisms of the Glyco-enzymes Associated with Pathogenesis of Klebsiella pneumoniae
Date Issued
2010
Date
2010
Author(s)
Chen, Ying-Yin
Abstract
The Gram-negative bacterium, Klebsiella pneumoniae (Kp), is an opportunistic pathogen that mainly causes primary pyogenic liver abscess, metastatic meningitis, sepsis and endophthalmitis in Taiwan. The main thrust of this thesis work is the understanding of the reaction mechanisms of two target proteins associated with the pathogenesis of K. pneumoniae. One is 6-phosphogluconate dehydrogenase (6PGDH) that acts as a new cell wall adhesin in Streptococcus pneumoniae and Streptococcus suis, as well as the third enzyme of the pentose phosphate pathway, catalyzing the oxidative decarboxylation of 6-phosphogluconate to form ribulose 5-phosphate, along with the reduction of NADP+ to NADPH. The other is UDP-glucose dehydrogenase (Ugd), catalyzing the NAD+-dependent 2-fold oxidation of UDP-glucose (UPG) to produce UDP-glucuronic acid (UGA), a requisite precursor for the biosynthesis of 4-amino-4-deoxy-L-arabinopyranose (L-Ara4N), that allows K. pneumoniae to resist the antibiotic polymyxin and protect gram-negative bacteria from the bactericidal action of cationic antimicrobial peptides (CAMPs) by the cationic modification of phosphates of lipid A with L-Ara4N. Here we report the first apo-form crystal structure of the pathogenic K. pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH, and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10° rotation and resulting in a 7 Å movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme. For 6PGDH, we propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits and the novel glycoconjugate-binding ability of 6PGDH provide important implications for the design of selective inhibitors. For K. pneumoniae Ugd (KpUgd), we have determined crystal structures of KpUgd in the apo and C-terminal 6×histidine-tagged states at high resolution as well as complexes with substrate, substrate/NADH, and two product molecules. The binding of two UGA molecules to the KpUgd structure reveals for the first time that the second UGA occupied the pre-existing position of the positively charged surface pocket distinct from the active site. Superimpositions and comparisons of our KpUgd structures indicated that the second UGA interacted with K256 and D257, which in turn gives rise to the concomitant movement of the substrate-interacting cysteine and forms an inactive configuration. The kinetic studies showed that KpUgd exhibited allosteric effects and the competitive inhibition of product implicated that the catalysis follows a negative feedback mechanism.
Subjects
Klebsiella pneumoniae
6-phosphogluconate dehydrogenase
UDP-glucose dehydrogenase
drug target
antibacterial resistance
Type
thesis
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