Assessment of the role of an omega loop of cholesterol oxidase: a truncated loop mutant has altered substrate specificity

Biochemistry
Nicole S SampsonK B Ghoshroy

Abstract

The function of an active site loop (70-90) of cholesterol oxidase has been ascertained by deleting five contiguous residues (79-83) from the tip of the loop. From the crystal structure of the wild-type enzyme, it appears that this truncation will not significantly perturb the structure of the rest of the enzyme. The UV/vis and CD spectra of the mutant confirm that the enzyme is properly folded with FAD bound. The mutant enzyme still transfers 2H from the 4beta-carbon of the intermediate, cholest-5-en-3-one, to the 6beta-carbon of the product, cholest-4-en-3-one, during isomerization. The kcat/Km of the mutant is increased 6-fold with dehydroepiandrosterone as substrate. Thus, the enzyme is still catalytically active after deletion of the five loop-tip residues. With micellar cholesterol, the kcat/Km of the mutant is decreased 170-fold relative to wild type. This suggests that the tip of the loop is necessary for packing with the "tail" of cholesterol and is responsible for substrate specificity at C17. Increased release of intermediate cholest-5-en-3-one in the mutant-catalyzed reaction is not observed. Truncation of the loop, therefore, does not affect the grip of the enzyme on the intermediate. With lipid vesicle substrates ...Continue Reading

Citations

Oct 6, 1999·Clinica Chimica Acta; International Journal of Clinical Chemistry·Y Nishiya, N Hirayama
Jan 5, 1999·Bioorganic & Medicinal Chemistry Letters·I J Kass, N S Sampson
Jul 17, 2007·Biochemistry·Xinxin YangNicole S Sampson
Feb 18, 2014·Acta Crystallographica. Section D, Biological Crystallography·Francesca SalviGiovanni Gadda
Dec 8, 2007·Biosensors & Bioelectronics·Sunil K AryaBansi D Malhotra
Feb 24, 2006·Proteins·Trevor P Creamer, Jacquelyn S Fetrow
Jun 10, 2010·The FEBS Journal·Oliver SpadiutChristina Divne
Oct 22, 2009·The FEBS Journal·Loredano PollegioniGianluca Molla
Oct 22, 2009·The FEBS Journal·Joseph Kreit, Nicole S Sampson
Oct 22, 2009·The FEBS Journal·Alice Vrielink, Sandro Ghisla
Jun 12, 2013·Colloids and Surfaces. B, Biointerfaces·Rubens A SilvaDenise F S Petri

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.