Procatalytic Ligand Strain. Ionization and Perturbation of 8-Nitroxanthine at the Urate Oxidase Active Site

The binding of the inhibitor 8-nitroxanthine to urate oxidase has been investigated by Raman and UV−visible absorption spectroscopy. The absorption maximum of 8-nitroxanthine shifts from 380 to 400 nm upon binding to the enzyme, demonstrating that the electronic structure of the ligand is perturbed. It has been proposed that oxidation of the substrate urate by urate oxidase is facilitated by formation of the substrate dianion at the enzyme active site, and Raman spectra of urate oxidase-bound 8-nitroxanthine suggest that both the dianionic and monoanionic forms of the ligand are bound to the enzyme under conditions where in solution the monoanion is present exclusively. The C4−C5 stretching frequency appears as a relatively isolated vibrational mode in 8-nitroxanthine whose frequency shifts according to the protonation state of the purine ring. Identification of the C4−C5 stretching mode was confirmed using [4-13C]-8-nitroxanthine and ab initio calculation of the vibrational modes. Two peaks corresponding to the C4−C5 stretching mode were evident in spectra of enzyme-bound 8-nitroxanthine, at 1541 and 1486 cm-1. The higher frequency peak was assigned to monoanionic 8-nitroxanthine, and the low-frequency peak was assigned to dianionic 8-nitroxanthine. The C4−C5 stretching frequency for free monoanionic 8-nitroxanthine was at 1545 cm-1, indicating that the enzyme polarizes that bond when the ligand is bound. The C4−C5 stretching frequency in dianionic 8-nitroxanthine is also shifted by 4 cm-1 to lower frequency upon binding. For 8-nitroxanthine free in solution, the C4−C5 stretching frequency shifts to lower frequency upon deprotonation, and the absorption maximum in the UV−visible spectrum shifts to higher wavelength. The spectral shifts observed upon binding of 8-nitroxanthine to urate oxidase are consistent with increased anionic character of the ligand, which is expected to promote catalysis in the reaction with the natural substrate urate. In the Raman spectra of 8-nitroxanthine bound to the F179A, F179Y, and K9M mutant proteins, the C4−C5 stretching frequency was not perturbed from its position for the unbound ligand. Both Vmax and V/K were decreased in the mutant enzymes, demonstrating a correlation between the interaction that perturbs the C4−C5 stretching frequency and the catalytic activity of the enzyme. It is suggested that hydrogen-bonding interactions that lead to precise positioning and deprotonation of the substrate are perturbed by the mutations

Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9α-Hydroxylase Enzymes

Rieske nonheme monooxygenase 3-ketosteroid 9α-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshAH37Rv of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a β sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the β sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 β-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities

Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase hisI

10.1021/bi050472wBiochemistry443010071-10080BICH