scholarly journals Probing the Role of Sigma π Interaction and Energetics in the Catalytic Efficiency of Endo-1,4-β-Xylanase

2012 ◽  
Vol 78 (24) ◽  
pp. 8817-8821 ◽  
Author(s):  
Raushan Kumar Singh ◽  
Manish Kumar Tiwari ◽  
In-Won Kim ◽  
Zhilei Chen ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acid ◽  
Turnover Rate ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Wild Type ◽  
High Turnover ◽  
Substrate Binding Pocket ◽  
Content Type ◽  
Important Residue

ABSTRACTChaetomium globosumendo-1,4-β-xylanase (XylCg) is distinguished from other xylanases by its high turnover rate (1,860 s−1), the highest ever reported for fungal xylanases. One conserved amino acid, W48, in the substrate binding pocket of wild-type XylCg was identified as an important residue affecting XylCg's catalytic efficiency.

scholarly journals Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

2010 ◽  
Vol 76 (5) ◽  
pp. 1653-1660 ◽  
Author(s):  
Ponnandy Prabhu ◽  
Marimuthu Jeya ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Catalytic Efficiency ◽  
Homology Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
High Turnover ◽  
Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

scholarly journals Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity

2004 ◽  
Vol 378 (2) ◽  
pp. 687-692 ◽  
Author(s):  
Carrie TSOI ◽  
Mikael WIDERSTEN ◽  
Ralf MORGENSTERN ◽  
Stellan SWEDMARK
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Fold Increase ◽  
Binding Pocket ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Substrate Binding Pocket ◽  
Site Selectivity ◽  
Km Value ◽  
Exogenous Compounds

The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure–activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146→Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in Km value) but 54-fold less efficient in sulphating dopamine (8-fold increase in Km value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.

scholarly journals Aromatic Amino Acid Mutagenesis at the Substrate Binding Pocket ofYarrowia lipolyticaLipase Lip2 Affects Its Activity and Thermostability

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Guilong Wang ◽  
Zimin Liu ◽  
Li Xu ◽  
Yunjun Yan
Keyword(s):  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Enzymatic Properties ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Substrate Binding Pocket ◽  
Significant Difference ◽  
Amino Acid Mutations

The lipase2 fromYarrowia lipolytica(YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towardsp-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted fromp-nitrophenyl laurate (pNPC12) top-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.

Role of the non-conserved amino acid asparagine 285 in the glycone-binding pocket of Neosartorya fischeri β-glucosidase

RSC Advances ◽  
2016 ◽  
Vol 6 (53) ◽  
pp. 48137-48144 ◽  
Author(s):  
Priyadharshini Ramachandran ◽  
Sujit Sadashiv Jagtap ◽  
Sanjay K. S. Patel ◽  
Jinglin Li ◽  
Yun Chan Kang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Pocket ◽  
High Turnover ◽  
Neosartorya Fischeri

Neosartorya fischeriβ-glucosidase (NfBGL595) is distinguished from other BGLs by its high turnover forp-nitrophenyl β-d-glucopyranoside (pNPG) and flavones.

scholarly journals Site-directed mutagenesis of mouse steroid 7α-hydroxylase (cytochrome P-4507α): role of residue-209 in determining steroid-cytochrome P-450 interaction

1993 ◽  
Vol 291 (2) ◽  
pp. 569-573 ◽  
Author(s):  
M Iwasaki ◽  
R L P Lindberg ◽  
R O Juvonen ◽  
M Negishi
Keyword(s):  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Hydroxylase Activity ◽  
Steroid Hydroxylase ◽  
Kd Values ◽  
Important Residue ◽  
Steroid Binding ◽  
Cytochrome P 450

We have cloned a cDNA encoding mouse steroid 7 alpha-hydroxylase P450(7) alpha (cytochrome P-450(7) alpha) and expressed it in Saccharomyces cerevisiae. Mouse P450(7) alpha is 70% identical in its amino acid sequence with the mouse steroid 15 alpha-hydroxylase P450(15) alpha (2A4). The Leu at position 209 of P450(15) alpha is the most important residue to determine the steroid hydroxylase activity of the P450 [Lindberg and Negishi (1989) Nature (London) 339, 632-634]. The P450(7) alpha contains Asn at the position corresponding to the Leu-209 of P450(15) alpha, although both P450s hydroxylate testosterone. The CO-reduced P450(7) alpha complex is unstable, so that it is quickly converted into the inactive P420, whereas the P450(15) alpha is very stable. The P450(7) alpha, however, is stabilized either by addition of testosterone or by a mutation of Asn-209 to Leu. The mutant P450(7) alpha displays a 17-fold lower Vmax. value than the wild-type enzyme. Unexpectedly, it also has 3-fold lower Km and Kd values. Residue 209 in P450(7) alpha, therefore, appears to be located at a critical site of the haem-substrate-binding pocket. Corticosterone inhibits the testosterone 7 alpha-hydroxylase activity of the wild-type P450(7) alpha, whereas it does not inhibit the mutant P450(7) alpha. Conversely, the P450(15) alpha activity becomes inhibited by corticosterone upon the replacement of Leu-209 by Asn. In addition, this mutation increases the corticosterone 15 alpha-hydroxylase activity of P450(15) alpha at least 20-fold. Whereas the inhibition by corticosterone depends on the presence of Asn at position 209, deoxycorticosterone inhibits the activities of the P450s regardless of the type of residue at 209. The results indicate, therefore, that the identity of residue 209 determines the affinity as well as specificity of steroid binding to both P450(7) alpha and P450(15) alpha.

scholarly journals Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

2013 ◽  
Vol 57 (10) ◽  
pp. 4990-4998 ◽  
Author(s):  
S. F. Queener ◽  
V. Cody ◽  
J. Pace ◽  
P. Torkelson ◽  
A. Gangjee
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Standard Therapy ◽  
Binding Pocket ◽  
Pneumocystis Jirovecii ◽  
Wild Type ◽  
Resistant Variant ◽  
Strong Activity ◽  
Content Type ◽  
Naturally Occurring

ABSTRACTPneumocystis jiroveciiis an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants ofP. jiroveciiDHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, withKivalues for trimethoprim 4-fold to 100-fold higher than those for the wild-typeP. jiroveciiDHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increasedKmvalues for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms ofP. jiroveciiDHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling ofkcat, as well as aKmfor NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR fromP. jiroveciiproduce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

scholarly journals Carbamate C-N Hydrolase Gene ameH Responsible for the Detoxification Step of Methomyl Degradation in Aminobacter aminovorans Strain MDW-2

2020 ◽  
Vol 87 (1) ◽  
Author(s):  
Wankui Jiang ◽  
Chenfei Zhang ◽  
Qinqin Gao ◽  
Mingliang Zhang ◽  
Jiguo Qiu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Degradation Mechanism ◽  
Catalytic Efficiency ◽  
Acid Sites ◽  
Amino Acid Residues ◽  
Content Type ◽  
Key Enzyme ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Methomyl {bis[1-methylthioacetaldehyde-O-(N-methylcarbamoyl)oximino]sulfide} is a highly toxic oxime carbamate insecticide. Several methomyl-degrading microorganisms have been reported so far, but the role of specific enzymes and genes in this process is still unexplored. In this study, a protein annotated as a carbamate C-N hydrolase was identified in the methomyl-degrading strain Aminobacter aminovorans MDW-2, and the encoding gene was termed ameH. A comparative analysis between the mass fingerprints of AmeH and deduced proteins of the strain MDW-2 genome revealed AmeH to be a key enzyme of the detoxification step of methomyl degradation. The results also demonstrated that AmeH was a functional homodimer with a subunit molecular mass of approximately 34 kDa and shared the highest identity (27%) with the putative formamidase from Schizosaccharomyces pombe ATCC 24843. AmeH displayed maximal enzymatic activity at 50°C and pH 8.5. Km and kcat of AmeH for methomyl were 87.5 μM and 345.2 s−1, respectively, and catalytic efficiency (kcat/Km) was 3.9 μM−1 s−1. Phylogenetic analysis revealed AmeH to be a member of the FmdA_AmdA superfamily. Additionally, five key amino acid residues (162, 164, 191, 193, and 207) of AmeH were identified by amino acid variations. IMPORTANCE Based on the structural characteristic, carbamate insecticides can be classified into oxime carbamates (methomyl, aldicarb, oxamyl, etc.) and N-methyl carbamates (carbaryl, carbofuran, isoprocarb, etc.). So far, research on the degradation of carbamate pesticides has mainly focused on the detoxification step and hydrolysis of their carbamate bond. Several genes, such as cehA, mcbA, cahA, and mcd, and their encoding enzymes have also been reported to be involved in the detoxification step. However, none of these enzymes can hydrolyze methomyl. In this study, a carbamate C-N hydrolase gene, ameH, responsible for the detoxification step of methomyl in strain MDW-2 was cloned and the key amino acid sites of AmeH were investigated. These findings provide insight into the microbial degradation mechanism of methomyl.

scholarly journals Role of a Conserved Membrane Glycine Residue in a Dicarboxylate Transporter from Sinorhizobium meliloti

2006 ◽  
Vol 189 (5) ◽  
pp. 2160-2163 ◽  
Author(s):  
Maria A. Trainer ◽  
Svetlana N. Yurgel ◽  
Michael L. Kahn
Keyword(s):  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Significant Activity ◽  
Nitrogen Fixing ◽  
Substrate Binding Pocket ◽  
Glycine Residue

ABSTRACT Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

scholarly journals Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Rie Ohara ◽  
Marilyn J. Roossinck
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Viral Genome ◽  
Large Scale ◽  
Viral Rna ◽  
Wild Type ◽  
Competitive Fitness ◽  
Content Type ◽  
Synonymous Substitutions ◽  
Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

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