Search

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,β-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,β-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification. [ABSTRACT FROM AUTHOR]

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4.hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to [H.sub.2][O.sub.2] and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

Di- and triketone inhibitors of (4-hydroxyphenyl) pyruvate dioxygenase (HPPD) are both effective herbicides and therapeutics. From this structure it is suggested that the interaction of triketone inhibitors with HPPD is topologically conserved and spices selectivity must therefore arise from differences in bioavailability.

Position 5 of the indole ring of tryptophan appears to play an important role in the formation of 5-hydroxytryptophan from tryptophan catalyzed by tryptophan hydroxylase. Oxygen transfer to the amino acid seems to be the rate-limiting step in the reaction.

The role of the serine hydrogen bond in the function of the native p-hydroxybenzoate hydroxylase (PHBH) has been studied by mutating residue 212 to alanine. PHBH is an aromatic flavoprotein hydroxylase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Results indicate that the residue is involved in substrate binding. The dissociation constant for p-hydroxybenzoate is one order of magnitude higher than for the wild-type but the catalytic process remains the same.

The enzyme p-hydroxybenzoate hydroxylase (PHBH) has to bind an ionizable phenolic substrate so that the NADPH may effectively reduce the enzyme's flavin. In the presence of NADPH, ionization of the phenol constitutes a 'password' that allows movement of the flavin ring system toward the surface of the enzyme, where reduction takes place. The linkage of substrate deprotonation to flavin movement is a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.

A study mutated lysine 297 of p-hydroxybenzoate hydroxylase to the neutral methionine residue and characterized the properties of the mutant enzyme. Findings indicate that regions of positive potential in the active site facilitate an important substrate activation that is required for rapid hydroxylation. They are consistent with existing theories of flavin mobility and substrate activation and underscore the significance of charge in the function of p-hydroxybenzoate hydroxylase.

Mutations at position 220 change the stability of the conformational states of the flavin of p-hydroxybenzoate hydroxylase and thus lead to changes in enzyme catalysis. The R220K enzyme stabilizes the conformation with flavin 'out' more than the wild type (WT) enzyme does, which lowers the R220K enzyme's catalytic efficiency. Catalytic mechanisms of the mutant R220K enzyme and the WT enzymes differ, as with partial hydroxylation of pOHB by R220K.