Genomic heterogeneity within conserved metabolic pathways of Arthrobacter species - a bioinformatic approach

Pal, Ayon; Mondal, Uttam Kumar; Mukhopadhyay, Soumik; Bothra, Asim K.

doi:10.6026/97320630005446

Cited by 3 publications

(4 citation statements)

References 21 publications

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“…Enzymes with higher thermostability are often observed to have a higher soluble expression level, as less stable proteins have shorter lifetimes and do not accumulate in the cytosol to high levels. , The relatively small difference in solubility in this instance may be due to the poor stability of apo-TrzN in E. coli , which is a bottleneck in the accumulation of soluble holo-TrzN . This is thought to be partly due to differences in codon usage, metabolism, and the chaperones present in E. coli and the native host ( Arthobacter and Nocardioides ). , The increased soluble expression level of of Gln241-TrzN, while small, does represent a physiologically relevant trade-off between a poorer catalyst that can reach a higher effective concentration in the cell (Gln241-TrzN) and a catalytically superior TrzN with a lower effective concentration (Glu241-TrzN). ,− …”

Section: Resultsmentioning

confidence: 99%

“…37 This is thought to be partly due to differences in codon usage, metabolism, and the chaperones present in E. coli and the native host (Arthobacter and Nocardioides). 68,69 The increased soluble expression level of of Gln241-TrzN, while small, does represent a physiologically relevant trade-off between a poorer catalyst that can reach a higher effective concentration in the cell (Gln241-TrzN) and a catalytically superior TrzN with a lower effective concentration (Glu241-TrzN). 66,70−73 Structural Characterization of TrzN Variants.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases

et al. 2016

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The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pK of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases

et al. 2016

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“…27 Eleven P450s with 490% sequence identity and a further nineteen with 460% sequence identity were discovered in different strains of Bacillus bacteria. A much larger number, with 440% sequence identity, were found (B200) in strains of Bacillus and related bacteria such as Paenibacillus, 28 Brevibacillus 29 and other bacterial strains such as Arthrobacter, 30 Ktedonobacter, 31 Herpetosiphon, 32 Haloferax 33 and S. cellulosum (CYP109C1, CYP109C2 and CYP109D1). The CYP109B1 enzyme also shares 440% sequence identity with the CYP106A1 and CYP106A2 enzymes, both from strains of B. megaterium.…”

Section: Phylogenetic Analysis Of the Cyp109 Familymentioning

confidence: 99%

The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 from Bacillus subtilis

Zhang

Hall

et al. 2015

Mol. BioSyst.

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The crystal structure of the versatile CYP109B1 enzyme from Bacillus subtilis has been solved at 1.8 Å resolution. This is the first structure of an enzyme from this CYP family, whose members are prevalent across diverse species of bacteria. In the crystal structure the enzyme has an open conformation with an access channel leading from the heme to the surface. The substrate-free structure reveals the location of the key residues in the active site that are responsible for binding the substrate in the correct orientation for regioselective oxidation. Importantly, there are significant differences among these residues in members of the CYP109 and closely related CYP106 families and these likely account for the variations in substrate binding and oxidation profiles observed with these enzymes. A whole-cell oxidation biosystem was developed, which contains CYP109B1 and a phthalate family oxygenase reductase (PFOR), from Pseudomonas putida KT24440, as the electron transfer partner. This electron transfer system is able to support CYP109B1 activity resulting in the regioselective hydroxylation of both α- and β-ionone in vivo and in vitro. The PFOR is therefore a versatile electron transfer partner that is able to support the activity of CYP enzymes from other bacterium. The crystal structure of CYP109B1 has a positively charged proximal face and this explains why it can interact with PFOR and adrenodoxin which are predominantly negatively charged around their [2Fe-2S] clusters.

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“…It might have taken place as a result of their early advent in evolutionary history as well as the process possesses high efficiency. One thus looks for an opportunity for carrying out comparative analysis of metabolic pathways among widely diverse species with an aim to extract information about the functional relation of organisms [1, 2]. The pentose phosphate pathway or hexose monophosphate shunt exemplifies one such important metabolic pathway which meets the need of all organisms for providing reducing power in the form of NADPH to execute anabolism.…”

Section: Introductionmentioning

confidence: 99%

Statistical analysis of pentose phosphate pathway genes from eubacteria and eukarya reveals translational selection as a major force in shaping codon usage pattern

Pal

Mukhopadhyay²,

Bothra³

2013

Bioinformation

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Comparative analysis of metabolic pathways among widely diverse species provides an excellent opportunity to extract information about the functional relation of organisms and pentose phosphate pathway exemplifies one such pathway. A comparative codon usage analysis of the pentose phosphate pathway genes of a diverse group of organisms representing different niches and the related factors affecting codon usage with special reference to the major forces influencing codon usage patterns was carried out. It was observed that organism specific codon usage bias percolates into vital metabolic pathway genes irrespective of their near universality. A clear distinction in the codon usage pattern of gram positive and gram negative bacteria, which is a major classification criterion for bacteria, in terms of pentose phosphate pathway was an important observation of this study. The codon utilization scheme in all the organisms indicates the presence of translation selection as a major force in shaping codon usage. Another key observation was the segregation of the H. sapiens genes as a separate cluster by correspondence analysis, which is primarily attributed to the different codon usage pattern in this genus along with its longer gene lengths. We have also analyzed the amino acid distribution comparison of transketolase protein primary structures among all the organisms and found that there is a certain degree of predictability in the composition profile except in A. fumigatus and H. sapiens, where few exceptions are prominent. In A. fumigatus, a human pathogen responsible for invasive aspergillosis, a significantly different codon usage pattern, which finally translated into its amino acid composition model portraying a unique profile in a key pentose phosphate pathway enzyme transketolase was observed.

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Genomic heterogeneity within conserved metabolic pathways of Arthrobacter species - a bioinformatic approach

Cited by 3 publications

References 21 publications

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases

The crystal structure of the versatile cytochrome P450 enzyme CYP109B1 from Bacillus subtilis

Statistical analysis of pentose phosphate pathway genes from eubacteria and eukarya reveals translational selection as a major force in shaping codon usage pattern

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