74784-45-5Relevant academic research and scientific papers
Biosynthesis of acaterin: Mechanism of the reaction catalyzed by dehydroacaterin reductase
Nakano, Sayaka,Sakane, Wataru,Oinaka, Hiroshi,Fujimoto, Yoshinori
, p. 6404 - 6408 (2006)
Dehydroacaterin reductase is an enzyme which catalyzes the final step of acaterin biosynthesis, that is, the reduction of the C-4/C-5 double bond of dehydroacaterin. The mechanism of the reduction was investigated with a cell-free preparation obtained from the acaterin-producing microorganism, Pseudomonas sp. A 92. Incubation of dehydroacaterin in the presence of [4,4-2H2]NADPH or D2O followed by 2H NMR analysis of the resulting acaterin revealed that the deuterium atom from NADPH was incorporated into the C-5 position of acaterin, while the deuterium atom from D2O was introduced into the C-4 position. It was further demonstrated that the pro-R hydrogen at C-4 of NADPH was stereospecifically utilized in this reduction.
α-secondary isotope effects as probes of "tunneling-ready" configurations in enzymatic H-tunneling: Insight from environmentally coupled tunneling models
Pudney, Christopher R.,Hay, Sam,Sutcliffe, Michael J.,Scrutton, Nigel S.
, p. 14053 - 14058 (2006)
Using α-secondary kinetic isotope effects (2° KIEs) in conjunction with primary (1°) KIEs, we have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate reductase (PETNR). We find exalted 2° KIEs (1.17-1.18) for both enzymes, consistent with hydrogen tunneling. These 2° KIEs, unlike 1° KIEs, are independent of promoting motions-a nonequilibrium pre-organization of cofactor and active site residues that is required to bring the reactants into a "tunneling-ready" configuration. That these 2° KIEs are identical suggests the geometries of the "tunneling-ready" configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a clearly temperature-dependent 1° KIE. The work emphasizes the benefit of combining studies of 1° and 2° KIEs to report on pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling.
Stereospecificity of hydride transfer for the catalytically recycled NAD+ in CDP-D-glucose 4,6-dehydratase
Hallis, Tina M.,Liu, Hung-Wen
, p. 15975 - 15982 (1998)
CDP-D-glucose 4,6-dehydratase (E(od)), found in the biosynthetic pathway of 3,6-dideoxysugars, contains a tightly bound MAD+ that is recycled during catalysis. The stereochemical preference of the hydride transfer to and from the coenzyme in E(od) was determined to be pro-S by analyzing the NAD+ produced when the apoenzyme was incubated with stereospecifically labeled NADH and its product, CDP-6-deoxy-D-glycero-L-threo-4-hexulose.
Substrate selectivity of an isolated enoyl reductase catalytic domain from an iterative highly reducing fungal polyketide synthase reveals key components of programming
Roberts, Douglas M.,Bartel, Christoph,Scott, Alan,Ivison, David,Simpson, Thomas J.,Cox, Russell J.
, p. 1116 - 1126 (2017/02/10)
A cis-acting enoyl reductase (ER) catalytic domain was isolated from a fungal highly reducing iterative polyketide synthase (HR-iPKS) for the first time and studied in vitro. The ER from the squalestatin tetraketide synthase forms a discrete dimeric protein in solution. The ER shows broad substrate selectivity, reducing enoyl species including both natural and unnatural substrates. Pantetheine-bound substrate thiolesters reacted much faster than the corresponding SNAC thiolesters. The unnatural substrates included Z-olefins, 2-ethyl olefins and pentaketides. Methylation of the substrate modifies the activity of the ER such that the 2,4-dimethyl oct-2-enoyl substrate fits into the active site but cannot be reduced. A new NMR-based assay was developed for the direct observation of the stereochemical preferences at the 4′ position of the NADPH cofactor and the C-2 and C-3 positions of the substrates. The assay reveals that the fungal iPKS ER-catalysed reaction is stereochemically identical to that of the vertebrate FAS (vFAS) at the cofactor 4′ position and the substrate 3-position, but the high stereoselectivity displayed by intact SQTKS is lost such that reprotonation at the 2-position is unselective by the isolated ER. A 3D model of ER was consistent with these observations and showed that the ER may sequester its final substrate to prevent further chain extension. The results support a developing model for programming by HR-iPKS in which competition for substrates between restrictive and permissive catalytic domains chaperones the growing polyketide to completion, while allowing for errors and evolution.
Kinetic and solvent isotope effects on biotransformation of aromatic amino acids and their derivatives
Kańska, Marianna,Jemielity, Jacek,Paj?k, Ma?gorzata,Pa?ka, Katarzyna,Podsadni, Katarzyna,Winnicka, El?bieta
, p. 627 - 634 (2016/12/26)
Aromatic amino acids such as l-phenylalanine, l-tryptophan, 3′,4′-dihydroxy-l-phenylalanine (l-DOPA), and their derivatives 3′,4′-dihydroxyphenylacelaldehyde (DOPAL) and 3′,4′-dihydroxyphenylethanol (DOPET), play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these metabolites draw biochemists' and medical researchers' attention. This study investigates the mechanisms of biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation of l-phenylalanine, 5′-chloro-l-tryptophan, and l-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the deamination of 2′-chloro-l-phenylalanine in the presence of the enzyme l-phenylalanine dehydrogenase, as well as in the conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard) radiometric methods.
Site-saturation mutagenesis of tryptophan 116 of saccharomyces pastorianus old yellow enzyme uncovers stereocomplementary variants
Padhi, Santosh Kumar,Bougioukou, Despina J.,Stewart, Jon D.
experimental part, p. 3271 - 3280 (2009/07/30)
Site-saturation mutagenesis was used to generate all possible replacements for Trp 116 of Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis) old yellow enzyme (OYE). Our original hypothesissthat smaller amino acids at position 116 would allow better acceptance of bulky 3-alkylsubstituted 2-cyclohexenonessproved incorrect. Instead, Phe and Ile replacements favored the binding of some substrates in an opposite orientation, which yielded reversed stereochemical outcomes compared to that of the wild-type OYE. For example, W116I OYE reduced (R)- and (S)-carvone to enantiomeric products, rather than the diastereomers produced by the wild-type OYE. Deuterium labeling revealed that (S)-carvone reduction by the W116I OYE occurred by the same pathway as that by the wild type (net trans-addition of H2), proving that different substrate binding orientations were responsible for the divergent products. Trp 116 mutants also afforded different stereochemical outcomes for reductions of (R)- perillaldehyde and neral. Preliminary studies of an OYE family member whosenative sequence contains Ile at position 116 (Pichia stipitis OYE 2.6) revealed that this enzyme's stereoselectivity matched that of the wild-t ype S. pastorianus OYE, showing that the identity of the residue at position 116 does not solely determine the substrate binding orientation. Computational docking studies using an induced fit methodology successfully reproduced the majority of the experimental outcomes. These computational tools will allow preliminary in silico screening of additional residues to identify those most likely to control the substrate binding orientation and provide some guidance to future experimental studies.
New insights into the mechanism of CDP-D-tyvelose 2-epimerase: An enzyme-catalyzing epimerization at an unactivated stereocenter
Hallis,Zhao,Liu
, p. 10493 - 10503 (2007/10/03)
Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of Yersinia pseudotuberculosis IVA and is the only member of this class of sugars to be produced directly from another 3,6-dideoxyhexose, paratose. The C-2 epimerization required for this conversion h
Melanin Biosynthesis: A Study of Polyphenol Deoxygenation
Viviani, Fabrice,Gaudry, Michel,Marquet, Andree
, p. 1255 - 1259 (2007/10/02)
The 1,3,6,8-Tetrahydroxynaphthalene (T4HN) reductase of Verticillium dahliae has been studied in a cell-free system.The use of specifically labelled 4(R)- and NADPH in the reduction of T4HN to scytalone reveals that the l
Stereochemistry of Reduction of the Vitamin Folic acid by Dihydrofolate Reductase
Charlton, Peter A.,Young, Douglas W.,Birdsall, Berry,Feeney, James,Roberts, Gordon C. K.
, p. 1349 - 1354 (2007/10/02)
Reduction of the vitamin folic acid (6) to the coenzyme 5,6,7,8-tetrahydrofolic acid (1) by the enzyme dihydrofolate reductase is shown to involve transfer of the 4-pro R hydrogen of NADPH to the same face at both C-6 and C-7 of the pteridine system (the re face at C-6 and the si face at C-7).The orientations of the pteridine system of folic acid (6) and of dihydrofolic acid (5) when bound to the enzyme are different from the orientation of the pteridine ring of the anti-cancer drug methotrexate (11) when bound to this enzyme.
