Amino Acid Residues Interacting with Both the Bound Quinone and Coenzyme, Pyrroloquinoline Quinone, in Escherichia coli Membrane-bound Glucose Dehydrogenase

Abstract

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e aq ؊ ) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e aq ؊ , although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) 2 belongs to the quinoprotein family with PQQ as a coenzyme (1, 2), and it catalyzes D-glucose oxidation to D-gluconate at the periplasmic side to transfer electrons to ubiquinol oxidase via UQ in the respiratory chain (3-5). Topological analysis revealed that mGDH consists of an N-terminal hydrophobic domain with five membrane-spanning segments and a large C-terminal domain residing in the periplasm, which contains PQQ and Ca 2ϩ -or Mg 2ϩ -binding sites in a superbarrel structure, conserved in quinoproteins (6 -8). Although its tertiary structure has not been resolved, the arrangement of amino acid residues around PQQ has been modeled on the basis of the crystal structure of the quinoprotein methanol dehydrogenase (6) as depicted in Fig. 1. The arrangement has been confirmed by results of several experiments with site-directed amino acid substitutions (9 -13). The orthoquinone portion of PQQ is a vital part for the catalytic reaction, to which Lys-493 hydr...

Menaquinone as Well as Ubiquinone as a Bound Quinone Crucial for Catalytic Activity and Intramolecular Electron Transfer in Escherichia coli Membrane-bound Glucose Dehydrogenase

“…The characteristics of the spectrum appear to consist of a broad signal with the peak-to-peak line width of nearly 100 G and a small signal at g ϭ 2.004 (at ϳ3400 G) with that of nearly 10 G (Ref. 25 and Fig. 4A).…”

“…Although its tertiary structure has not been determined, the arrangement of amino acid residues around PQQ has been modeled (19) and has been confirmed by results of several experiments with site-directed amino acid substitutions (20 -24). The substitution of either Lys-493, which is modeled to hydrogen-bond to an orthoquinone portion of PQQ as a vital part for the catalytic reaction, or Asp-466, which is located close to that portion and functions in extraction of a proton from substrate glucose, has a severe effect on the catalytic ability of the enzyme (24,25).…”

“…It has been demonstrated that mGDH has two UQ-binding sites, one (Q I ) for bound UQ and the other (Q II ) for bulk UQ (26), which is near the membrane surface rather than in the hydrophobic interior (27), and that intramolecular electron transfer following the catalytic reaction occurs from PQQH 2 directly to UQ in the Q II site or via bound UQ. Our recent studies revealed that e aq Ϫ produced by pulse radiolysis caused a rapid reduction of bound UQ followed by intramolecular electron transfer to PQQ in mGDH and suggested that the two redox centers are closely located at a distance of 11-13 Å (28) and that Asp-466 and Lys-493 are involved in proton donation to the semiquinone anion radical of bound UQ and in electron transfer from bound UQ to PQQ, respectively (25). However, no evidence for the interaction with MQ has so far been provided in mGDH.…”

Menaquinone as Well as Ubiquinone as a Crucial Component in the Escherichia coli Respiratory Chain

“…6). Pulse radiolysis analysis revealed that the two redox centers are closely located at a distance of 11-13 Å and that Asp466 and Lys493 are involved in proton donation to the semiquinone anion radical of bound Q and in electron transfer from bound UQ to PQQ, respectively (Elias et al, 2000;Mustafa et al, 2008a). Recent mGDH analysis provided the first evidence that the primary dehydrogenase in respiratory chains utilizes both MK and UQ as a bound Q and suggest that bound MK occurs in a fashion similar to that of bound UQ in the mGDH molecule and functions as an electron acceptor from PQQ (Mustafa et al, 2008b).…”

The Life History of Pyrroloquinoline Quinone (PQQ): A Versatile Molecule with Novel Impacts on Living Systems

“…PQQ prevents accumulation of alpha-synuclein and amyloid beta proteins [103]. PQQ provides advanced level of protection on endothelial cells of mouse brain from Gluco-damage by suppression of ROS and cell death by blocking signaling pathway of JNK [104][105][106][107].The treatment of proliferated Schwann cells with various concentrations of PQQ enhanced the expression of CREB, c-fos, c-jun and PCNA [108]. PQQ on traumatic brain injury (TBI) has found to be neuroprotective [109] as measurements of physiological parameters indicated that PQQ restrains function of brain in older people related attention, working and memory [110].…”