Mechanistic studies on .beta.-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes

Biochemistry ◽  
1989 ◽  
Vol 28 (14) ◽  
pp. 5735-5742 ◽  
Author(s):  
Stewart Thompson ◽  
Friedrich Mayerl ◽  
Oliver P. Peoples ◽  
Satoru Masamune ◽  
Anthony J. Sinskey ◽  
...  
Keyword(s):  
Active Site ◽  
Mechanistic Studies ◽  
Wild Type ◽  
Thermodynamic Characterization ◽  
Zoogloea Ramigera

scholarly journals Kinetic Mechanistic Studies of Wild-Type Leucine-Rich Repeat Kinase2: Characterization of the Kinase and GTPase Activities

Biochemistry ◽  
2010 ◽  
Vol 49 (9) ◽  
pp. 2008-2017 ◽  
Author(s):  
Min Liu ◽  
Brittany Dobson ◽  
Marcie A. Glicksman ◽  
Zhenyu Yue ◽  
Ross L. Stein
Keyword(s):  
Leucine Rich Repeat ◽  
Mechanistic Studies ◽  
Wild Type

scholarly journals Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

1990 ◽  
Vol 271 (2) ◽  
pp. 487-491 ◽  
Author(s):  
A Hädener ◽  
P R Alefounder ◽  
G J Hart ◽  
C Abell ◽  
A R Battersby
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Kinetic Studies ◽  
Enzyme Kinetic ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Over Expression ◽  
Putative Active Site ◽  
Lysine Residues

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5′-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

scholarly journals Identification and Structural Characterization of I84C and I84A Mutations That Are Associated with High-Level Resistance to Human Immunodeficiency Virus Protease Inhibitors and Impair Viral Replication

2006 ◽  
Vol 51 (2) ◽  
pp. 732-735 ◽  
Author(s):  
Hongmei Mo ◽  
Neil Parkin ◽  
Kent D. Stewart ◽  
Liangjun Lu ◽  
Tatyana Dekhtyar ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Active Site ◽  
Accessible Surface Area ◽  
Wild Type ◽  
Human Immunodeficiency Virus Protease ◽  
Immunodeficiency Virus ◽  
Accessible Surface ◽  
High Level ◽  
Level Resistance

ABSTRACT Two novel human immunodeficiency virus protease mutations, I84C and I84A, were identified in patient isolates. The mutants with I84C displayed high-level resistance (median, at least 56-fold) to nelfinavir and saquinavir, but the majority remained susceptible to lopinavir. In contrast, isolates with the I84A mutation exhibited ≥33-fold median increased levels of resistance to nelfinavir, indinavir, amprenavir, ritonavir, lopinavir, saquinavir, and atazanavir. Isolates with the I84A or I84C mutation tended to be more resistant than the isolates with the I84V mutation. Modeling of the structure of the mutant proteases indicated that the I84V, I84C, and I84A mutations all create unoccupied volume in the active site, with I84A introducing the greatest change in the accessible surface area from that of the wild-type structure.

Characterization of wild-type and an active-site mutant in Escherichia coli of short-chain acyl-CoA dehydrogenase from Megasphaera elsdenii

Biochemistry ◽  
1993 ◽  
Vol 32 (40) ◽  
pp. 10736-10742 ◽  
Author(s):  
Donald F. Becker ◽  
James A. Fuchs ◽  
David K. Banfield ◽  
Walter D. Funk ◽  
Ross T. A. MacGillivray ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Short Chain ◽  
Wild Type ◽  
Megasphaera Elsdenii ◽  
Chain Acyl

scholarly journals Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1208 ◽  
Author(s):  
Mohamed H. Habib ◽  
Henriëtte J. Rozeboom ◽  
Marco W. Fraaije
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sequence Motif ◽  
Acidic Ph ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Values

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

scholarly journals Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Suprama Datta ◽  
Uday S. Annapure ◽  
David J. Timson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steric Hindrance ◽  
Active Site ◽  
Limited Range ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Aliphatic Aldehydes ◽  
Aldehyde Dehydrogenases ◽  
Equivalent Residue

Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met177, compared with the equivalent residue Leu196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity.

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