Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis.

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.

Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans.

The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H/C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k(lim)) for TTQ reduction is controlled by a single ionization with pK(a) value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k(lim)/K(d) of pK(a) values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential ionizations is discussed.

Translation initiation factors eIF4E and eIFiso4E are required for polysome formation and regulate plant growth in tobacco.

Eukaryotic initiation factor eIF4E plays a pivotal role in translation initiation. As a component of the ternary eIF4F complex, eIF4E interacts with the mRNA cap structure to facilitate recruitment of the 40S ribosomal subunit onto mRNA. Plants contain two distinct cap-binding proteins, eIF4E and eIFiso4E, that assemble into different eIF4F complexes. To study the functional roles of eIF4E and eIFiso4E in tobacco, we isolated two corresponding cDNAs, NteIF4E1 and NteIFiso4E1, and used these to deplete cap-binding protein levels in planta by antisense downregulation. Antibodies raised against recombinant NteIF4E1 detected three distinct cap-binding proteins in tobacco leaf extracts; NteIF4E and two isoforms of NteIFiso4E. The three cap-binding proteins were immuno-detected in all tissues analysed and were coordinately regulated, with peak expression in anthers and pollen. Transgenic tobacco plants showing significant depletion of either NteIF4E or the two NteIFiso4E isoforms displayed normal vegetative development and were fully fertile. Interestingly, NteIFiso4E depletion resulted in a compensatory increase in NteIF4E levels, whereas the down-regulation of NteIF4E did not trigger a reciprocal increase in NteIFiso4E levels. The antisense depletion of both NteIF4E and NteIFiso4E resulted in plants with a semi-dwarf phenotype and an overall reduction in polyribosome loading, demonstrating that both eIF4E and eIFiso4E support translation initiation in planta, which suggests their potential role in the regulation of plant growth.