Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.
Acta Crystallographica Section D Biological Crystallography (Impact Factor: 7.23). 08/2011; 67(Pt 8):671-7. DOI: 10.1107/S090744491101746X
Source: PubMed

ABSTRACT The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The stereospecific oxidative decomposition of urate into allantoin is the core of purine catabolism in many organisms. The spontaneous decomposition of upstream intermediates and the nonenzymatic racemization of allantoin lead to an accumulation of (R)-allantoin, because the enzymes converting allantoin into allantoate are specific for the (S) isomer. The enzyme allantoin racemase catalyzes the reversible conversion between the two allantoin enantiomers, thus ensuring the overall efficiency of the catabolic pathway and preventing allantoin accumulation. On the basis of recent crystallographic and biochemical evidence, allantoin racemase has been assigned to the family of cofactor-independent racemases, together with other amino acid racemases. A detailed computational investigation of allantoin racemase has been carried out to complement the available experimental data and to provide atomistic insight into the enzymatic action. Allantoin, the natural substrate of the enzyme, has been investigated at the quantum mechanical level, in order to rationalize its conformational and tautomeric equilibria, playing a key role in protein-ligand recognition and in the following catalytic steps. The reaction mechanism of the enzyme has been elucidated through quantum mechanics/molecular mechanics (QM/MM) calculations. The potential energy surface investigation, carried out at the QM/MM level, revealed a stepwise reaction mechanism. A pair of cysteine residues promotes the stereoinversion of a carbon atom of the ligand without the assistance of cofactors. Electrostatic fingerprint calculations are used to discuss the role of the active site residues in lowering the pKa of the substrate. The planar unprotonated intermediate is compared with the enolic allantoin tautomer observed in the active site of the crystallized enzyme. Finally, the enzymatic catalysis featured by allantoin racemase (AllR) is compared with that of other enzymes belonging to the same family.
    The Journal of Physical Chemistry B 06/2014; 118(27). DOI:10.1021/jp411786z · 3.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Often, we elucidate evolutionary processes backwards, starting with eutherian mammals and gradually climbing down the evolutionary tree to those species who have survived since long before mammals evolved. This is also true for elucidating the evolution of specific proteins, in this case, the protein currently known as "transthyretin" (TTR). TTR was first described in eutherian mammals and was known as a thyroxine (T4) binding protein. However, mammals are the exception among vertebrates in respect to the function of TTR, as in teleost fish, amphibians, reptiles and birds TTR preferentially binds triiodothyronine (T3), which is the active form of thyroid hormone (TH). The TTR gene possibly arose as a duplication of the transthyretin-like protein (TLP) gene, around the stage of the agnathans. Some vertebrate species have both the TTR and TLP genes, while others have "lost" the TLP gene. TLP genes have been found in all kingdoms. The TLPs analyzed to date do not bind THs or their analogs, but are enzymes involved in uric acid metabolism; specifically, they are 5-hydroxyisourate hydrolases. A Salmonella TLP knock-out strain demonstrated that TLP was essential for the bacteria's survival in the high uric acid environment of the chicken alimentary tract. Many other TLPs are yet to be characterized for their function although several have been confirmed as 5-hydroxyisourate hydrolases. This review describes the evolution of TLP/TTR and how subtle changes in gene structure or amino acid substitution can drastically change the function of this protein, without altering its overall 3D conformation.
    Frontiers in Endocrinology 01/2014; 5:245. DOI:10.3389/fendo.2014.00245
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated. © 2015 The Author. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
    Microbial Biotechnology 02/2015; 8(3). DOI:10.1111/1751-7915.12262 · 3.21 Impact Factor

Full-text (2 Sources)

Available from
May 20, 2014