8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

No abstract

The genes encoding the Escherichia coli flavodoxin NADP ϩ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a k cat of 1610.3 min Ϫ1 and a K m of 23.6 µM, and cytochrome c with a k cat of 141.3 min Ϫ1 and a K m of 17.6 µM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent K m of 6.84 µM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isolectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (Ϫ308 mV and Ϫ268 mV, respectively) and FLD (Ϫ254 mV and Ϫ433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH→FLDR→FLD. Binding of 2′ adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M Ϫ1 cm Ϫ1 at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.Keywords : flavodoxin ; flavodoxin NADP ϩ oxidoreductase; redox potentiometry; enzyme kinetics; cytochrome P-450.The Escherichia coli flavodoxin NADP ϩ oxidoreductase (FLDR or flavodoxin reductase) and flavodoxin (FLD) are the two flavin-containing components of a short electron-transfer chain from NADPH, which provides electrons for the function of the biotin synthase [1] and cobalamin-dependent methionine synthase systems [2]. The enzymes are also required during anaerobic growth of the organism, participating in the pyruvate formate/lyase system of E. coli Ϫ a crucial mechanism for the anaerobic generation of pyruvate for glycolysis [3] and in the generation of deoxyribonucleotides through the enzyme anaerobic ribonucleotide reductase [4]. Recently, the FLDR/FLD system has also been recognised as the E. coli 'reductase', which can support the functio...

In heterogeneous catalysis, the two main reaction mechanisms which have been proposed are the Langmuir–Hinshelwood and the Eley–Rideal. For the vast majority of surface catalytic reactions, it has been accepted that the Langmuir–Hinshelwood mechanism is preferred. In this study, we investigate catalytic CO oxidation on Pt(111). It is found that reaction barriers for Langmuir–Hinshelwood mechanisms actually tend to be higher than those for Eley–Rideal ones. An explanation is presented as to why it is still more probable for the reaction to proceed via the Langmuir–Hinshelwood mechanism, despite its higher reaction barrier.

No abstract