Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding.

The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 A. The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics.

Cofactor mimics as selective inhibitors of NAD-dependent inosine monophosphate dehydrogenase (IMPDH)--the major therapeutic target.

IMP dehydrogenase, the key enzyme in de novo synthesis of purine nucleotides, is an important therapeutic target. Three inhibitors of IMP dehydrogenase reached the market; ribavirin (Rebetol) a broad-spectrum antiviral agent, which in combination with interferon-alpha is now used for treatment of hepatitis C virus infections, mizoribine (Bredinin) and mycophenolic mofetil (CellCept) have been introduced as immunosuppressants. Numerous novel inhibitors are under development. This review describes recent progress in the development of new drugs based on inhibition of IMP dehydrogenase.

Crystallographic studies on the complex behavior of nicotine binding to P450cam (CYP101).

Crystallographic and spectroscopic studies have been undertaken to characterize the binding behavior of the non-native substrate nicotine in the active site of the monooxygenase hemoprotein cytochrome P450cam. Despite the existence of a theoretical model that is consistent with the observed distribution of monooxygenation products, the crystal structure of the complex indicates that the primary binding mode of nicotine is unproductive. The structure is confirmed by spectral data that indicate direct coordination of substrate pyridine nitrogen with the heme iron. This would be the proper structure for evaluating binding affinity and inhibition. Reduction of the heme from Fe(III) to Fe(II) and introduction of carbon monoxide into crystals of the nicotine-P450cam complex, to simulate molecular oxygen binding, produces reorientation of the nicotine. This orientation is the appropriate one for predicting regioselectivity and the kinetic features of substrate oxidation. While it is not clear that such complicated behavior will be exhibited for other enzyme-substrate interactions, it is clear that a single crystal structure for a given substrate-enzyme interaction may not provide a good description of the binding mode responsible for product formation.

The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol.

Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.

Novel mycophenolic adenine bis(phosphonate) analogues as potential differentiation agents against human leukemia.

Novel mycophenolic adenine dinucleotide (MAD) analogues have been prepared as potential inhibitors of inosine monophosphate dehydrogenase (IMPDH). MAD analogues resemble nicotinamide adenine dinucleotide binding at the cofactor binding domain of IMPDH; however, they cannot participate in hydride transfer and therefore inhibit the enzyme. The methylenebis(phosphonate) analogues C2-MAD and C4-MAD were obtained by coupling 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate) (22) with mycophenolic alcohols 20 and 21 in the presence of diisopropylcarbodiimide followed by deprotection. C2-MAD was also prepared by coupling of mycophenolic methylenebis(phosphonate) derivative 30 with 2',3'-O-isopropylideneadenosine. Compound 30 was conveniently synthesized by the treatment of benzyl-protected mycophenolic alcohol 27 with a commercially available methylenebis(phosphonic dichloride) under Yoshikawa's reaction conditions. C2-MAD and C4-MAD were found to inhibit the growth of K562 cells (IC(50) = 0.7 microM and IC(50) = 0.1 microM, respectively) as potently as mycophenolic acid (IC(50) = 0.3 microM). In addition, C2-MAD and C4-MAD triggered vigorous differentiation of K562 cells an order of magnitude more potently than tiazofurin, and MAD analogues were resistant to glucuronidation in vitro. These results show that C2-MAD and C4-MAD may be of therapeutic interest in the treatment of human leukemias.