scholarly journals An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17α-hydroxylase-17,20-lyase (P-45017α also CYP17)

1998 ◽  
Vol 330 (2) ◽  
pp. 967-974 ◽  
Author(s):  
Peter LEE-ROBICHAUD ◽  
E. Monika AKHTAR ◽  
Muhammad AKHTAR
Keyword(s):  
Hydrogen Bonds ◽  
Active Site ◽  
Catalytic Properties ◽  
Active Form ◽  
Proton Donor ◽  
Wild Type ◽  
Cleavage Reaction ◽  
Acyl Carbon ◽  
Hydroxylation Reaction

Certain cytochrome P-450s involved in the transformation of steroids catalyse not only the hydroxylation process associated with the group of enzymes, but also an acyl-carbon cleavage reaction. The hydroxylation occurs using an iron-monooxygen species while the acyl-carbon cleavage has been suggested to be promoted by an iron peroxide. In this paper we have studied the role of active site protic residues, Glu305 and Thr306, in modulating the two activities. For this purpose, the kinetic parameters for the hydroxylation reaction (pregnenolone → 17α-hydroxypregnenolone) and two different versions of acyl-carbon cleavage (17α-hydroxypregnenolone → dehydroepiandrosterone and 3β-hydroxyandrost-5-ene-17β-carbaldehyde → 3β-hydroxyandrost-5,16-diene+androst-5-ene-3β,17α-diol) were determined using the wild-type human CYP17 and its eight different single and double mutants. In addition the propensity of the proteins to undergo a subtle rearrangement converting the 450 nm active-form into an inactive counterpart absorbing at 420 nm, was monitored by measuring the of the P-450 → P-420 conversion. The results are interpreted to draw the following conclusions. The functional groups of Glu305 and Thr306 do not directly participate in the two proton delivery steps required for hydroxylation but may be important participants for the provision of a net work of hydrogen bonds for ‘activating’ water that then acts as a proton donor. The loss of any one of these residues is, therefore, only partially debilitating. That the mutation of Thr306 impairs the hydroxylation reaction more than it does the acyl-carbon cleavage is consistent with the detailed mechanistic scheme considered in this paper. Furthermore attention is drawn to the fact that the mutation of Glu305 and Thr306 subtly perturbed the architecture of the active site, which affects the geometry of this region of the protein and therefore its catalytic properties.

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

scholarly journals The Role of Vitamin D and Vitamin D Receptor in Immunity toLeishmania majorInfection

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
James P. Whitcomb ◽  
Mary DeAgostino ◽  
Mark Ballentine ◽  
Jun Fu ◽  
Martin Tenniswood ◽  
...  
Keyword(s):  
Vitamin D ◽  
Immune Responses ◽  
Vitamin D Receptor ◽  
Leishmania Major ◽  
Active Form ◽  
Wild Type ◽  
Vitamin D Signaling ◽  
Deficient Mice ◽  
Th 1

Vitamin D signaling modulates a variety of immune responses. Here, we assessed the role of vitamin D in immunity to experimental leishmaniasis infection in vitamin D receptor-deficient mice (VDRKO). We observed that VDRKO mice on a genetically resistant background have decreasedLeishmania major-induced lesion development compared to wild-type (WT) mice; additionally, parasite loads in infected dermis were significantly lower at the height of infection. Enzymatic depletion of the active form of vitamin D mimics the ablation of VDR resulting in an increased resistance toL. major. Conversely, VDRKO or vitamin D-deficient mice on the susceptible Th2-biased background had no change in susceptibility. These studies indicate vitamin D deficiency, either through the ablation of VDR or elimination of its ligand, 1,25D3, leads to an increase resistance toL. majorinfection but only in a host that is predisposed for Th-1 immune responses.

scholarly journals Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

1998 ◽  
Vol 180 (17) ◽  
pp. 4658-4666 ◽  
Author(s):  
Mary McLenigan ◽  
Thomas S. Peat ◽  
Ekaterina G. Frank ◽  
John P. McDonald ◽  
Martín Gonzalez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Potential Effect ◽  
Wild Type ◽  
Cleavage Reaction ◽  
Nonsense Mutations ◽  
Spontaneous Mutagenesis ◽  
Sos Mutagenesis ◽  
Low Copy Number ◽  
Induced Mutagenesis

ABSTRACT Although it has been 10 years since the discovery that theEscherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomalumuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.

scholarly journals The role of an active site Mg2+ in HDV ribozyme self-cleavage: insights from QM/MM calculations

2015 ◽  
Vol 17 (1) ◽  
pp. 670-679 ◽  
Author(s):  
Vojtěch Mlýnský ◽  
Nils G. Walter ◽  
Jiří Šponer ◽  
Michal Otyepka ◽  
Pavel Banáš
Keyword(s):  
Active Site ◽  
The Self ◽  
Direct Impact ◽  
Cleavage Reaction ◽  
Hdv Ribozyme ◽  
Specific Position

The specific position and coordination of active site Mg2+ ion have a significant direct impact on the self-cleavage reaction in HDV ribozyme.

scholarly journals Tailoring a Soluble Diiron Monooxygenase for Synthesis of Aromatic N-oxides

Catalysts ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 356 ◽  
Author(s):  
Vytautas Petkevičius ◽  
Justas Vaitekūnas ◽  
Dovydas Vaitkus ◽  
Narimantas Čėnas ◽  
Rolandas Meškys
Keyword(s):  
Organic Chemistry ◽  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Work Site ◽  
Whole Cells ◽  
New Variant

The aromatic N-oxides have received increased attention over the last few years due to their potential application in medicine, agriculture and organic chemistry. As a green alternative in their synthesis, the biocatalytic method employing whole cells of Escherichia coli bearing phenol monooxygenase like protein PmlABCDEF (from here on – PML monooxygenase) has been introduced. In this work, site-directed mutagenesis was used to study the contributions of active site neighboring residues I106, A113, G109, F181, F200, F209 to the regiospecificity of N-oxidation. Based on chromogenic indole oxidation screening, a collection of PML mutants with altered catalytic properties was created. Among the tested mutants, the A113G variant acquired the most distinguishable N-oxidations capacity. This new variant of PML was able to produce dioxides (quinoxaline-1,4-dioxide, 2,5-dimethylpyrazine-1,4-dioxide) and specific mono-N-oxides (2,3,5-trimethylpyrazine-1-oxide) that were unachievable using the wild type PML. This mutant also featured reshaped regioselectivity as N-oxidation shifted towards quinazoline-1-oxide compared to quinazoline-3-oxide that is produced by the wild type PML.

scholarly journals Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

2002 ◽  
Vol 68 (11) ◽  
pp. 5265-5273 ◽  
Author(s):  
Thomas J. Smith ◽  
Susan E. Slade ◽  
Nicolas P. Burton ◽  
J. Colin Murrell ◽  
Howard Dalton
Keyword(s):  
Protein Engineering ◽  
Active Site ◽  
Methane Monooxygenase ◽  
Expression System ◽  
Active Form ◽  
Wild Type ◽  
Active Site Residues ◽  
Α Subunit ◽  
Soluble Methane Monooxygenase ◽  
Type Activity

ABSTRACT Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O2- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

scholarly journals Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Martino L. Di Salvo ◽  
J. Neel Scarsdale ◽  
Galina Kazanina ◽  
Roberto Contestabile ◽  
Verne Schirch ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Serine Hydroxymethyltransferase ◽  
Active Site Residue ◽  
Wild Type ◽  
Hydroxymethyl Group ◽  
New Energy ◽  
Mutant Forms

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stablegem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion ofgem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and theε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

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