Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus

ABSTRACTA novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified inBacillus cereus. The amino acid sequence of ALDH fromB. cereus(BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)+. We newly found thatBcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues inBcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD+converted 400 μM all-trans-retinal to 210 μM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all-trans-retinal to 20, 90, and 40 μM all-trans-retinol, respectively. These results indicate thatBcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore,BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol.IMPORTANCEAlthough mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)+as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid byBcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 inBcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

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Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00711-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5678-5686 ◽

Cited By ~ 6

Author(s):

Mei Li ◽

Benjamin C. Conklin ◽

Magdalena A. Taracila ◽

Rebecca A. Hutton ◽

Marion J. Skalweit

Keyword(s):

Clavulanic Acid ◽

Susceptibility Testing ◽

Catalytic Efficiency ◽

Wild Type ◽

Oxyanion Hole ◽

Content Type ◽

Class A ◽

Inhibitor Resistance ◽

Structural Probes ◽

Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

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Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Applied and Environmental Microbiology ◽

10.1128/aem.07012-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 3880-3884 ◽

Cited By ~ 17

Author(s):

Yu-Ri Lim ◽

Soo-Jin Yeom ◽

Deok-Kun Oh

Keyword(s):

Specific Activity ◽

Thermus Thermophilus ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Geobacillus Thermodenitrificans ◽

Wild Type Enzyme ◽

Content Type ◽

Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

Applied and Environmental Microbiology ◽

10.1128/aem.01076-19 ◽

2019 ◽

Vol 85 (17) ◽

Cited By ~ 2

Author(s):

Zhu Jiang ◽

Tengfei Niu ◽

Xueqin Lv ◽

Yanfeng Liu ◽

Jianghua Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Catalytic Efficiency ◽

Molecular Engineering ◽

Fine Tuning ◽

Wild Type ◽

Content Type ◽

Chitosan Oligosaccharides ◽

In The Wild ◽

Secretory Production ◽

Extracellular Activity

ABSTRACT Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

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Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.06814-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2230-2240 ◽

Cited By ~ 40

Author(s):

Xiaoyun Su ◽

Roderick I. Mackie ◽

Isaac K. O. Cann

Keyword(s):

Catalytic Efficiency ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Wild Type ◽

Ph Optimum ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

Type Protein ◽

Wild Type Protein ◽

Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.00457-13 ◽

2013 ◽

Vol 79 (13) ◽

pp. 4072-4077 ◽

Cited By ~ 57

Author(s):

Xuguo Duan ◽

Jian Chen ◽

Jing Wu

Keyword(s):

Catalytic Efficiency ◽

Kinetic Studies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Ph Optimum ◽

Widespread Application ◽

Content Type ◽

Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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Vaccine Protection against Bacillus cereus-Mediated Respiratory Anthrax-Like Disease in Mice

Infection and Immunity ◽

10.1128/iai.01346-12 ◽

2013 ◽

Vol 81 (3) ◽

pp. 1008-1017 ◽

Cited By ~ 8

Author(s):

So-Young Oh ◽

Hannah Maier ◽

Jay Schroeder ◽

G. Stefan Richter ◽

Derek Elli ◽

...

Keyword(s):

Bacillus Cereus ◽

Neutralizing Antibodies ◽

Protective Antigen ◽

Fatal Case ◽

Virulence Plasmid ◽

Wild Type ◽

Lethal Challenge ◽

Vaccine Protection ◽

Content Type ◽

Virulence Plasmids

ABSTRACTBacillus cereusstrains harboring a pXO1-like virulence plasmid cause respiratory anthrax-like disease in humans, particularly in welders. We developed mouse models for intraperitoneal as well as aerosol challenge with spores ofB. cereusG9241, harboring pBCXO1 and pBC218 virulence plasmids. Compared to wild-typeB. cereusG9241, spores with a deletion of the pBCXO1-carried protective antigen gene (pagA1) were severely attenuated, whereas spores with a deletion of the pBC218-carried protective antigen homologue (pagA2) were not. Anthrax vaccine adsorbed (AVA) immunization raised antibodies that bound and neutralized thepagA1-encoded protective antigen (PA1) but not the PA2 orthologue encoded bypagA2. AVA immunization protected mice against a lethal challenge with spores fromB. cereusG9241 orB. cereusElc4, a strain that had been isolated from a fatal case of anthrax-like disease. As the pathogenesis ofB. cereusanthrax-like disease in mice is dependent onpagA1and PA-neutralizing antibodies provide protection, AVA immunization may also protect humans from respiratory anthrax-like death.

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Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants

Applied and Environmental Microbiology ◽

10.1128/aem.01013-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 1

Author(s):

Li Wang ◽

Chenchen Liang ◽

Jing Wu ◽

Liming Liu ◽

Keith E. J. Tyo

Keyword(s):

Protein Engineering ◽

Sulfolobus Solfataricus ◽

Catalytic Efficiency ◽

Wild Type ◽

Primer Extension Assay ◽

Base Pairs ◽

Content Type ◽

Thumb Domain ◽

Nucleotide Incorporation ◽

Incorporation Efficiency

ABSTRACT The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10−4 s−1 μM−1) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (k cat/Km ) at incorporating dCTP and dATP, respectively, than wild-type Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4. IMPORTANCE DNA polymerases with poor fidelity can be exploited to store data and record changes in response to the intracellular environment. Sulfolobus solfataricus Dpo4 is such an enzyme, although its use is hindered by its low processivity. In this work, we used a bioinformatics and protein engineering approach to generate Dpo4 mutants with improved processivity. We identified the Dpo4 thumb domain as the most relevant in controlling processivity.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02261-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8817-8821 ◽

Cited By ~ 7

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.

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Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Applied and Environmental Microbiology ◽

10.1128/aem.02624-16 ◽

2016 ◽

Vol 83 (2) ◽

Cited By ~ 10

Author(s):

Junxian Zheng ◽

Taowei Yang ◽

Junping Zhou ◽

Meijuan Xu ◽

Xian Zhang ◽

...

Keyword(s):

Disulfide Bonds ◽

Catalytic Efficiency ◽

Fold Increase ◽

Enzyme Inactivation ◽

Cysteine Residues ◽

Candida Boidinii ◽

Wild Type ◽

Nadh Regeneration ◽

Content Type ◽

The Cost

ABSTRACT NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10C fdh ) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10C fdh , which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances. IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

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Mutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-β-lactamase

Biochemical Journal ◽

10.1042/bj3630687 ◽

2002 ◽

Vol 363 (3) ◽

pp. 687-696 ◽

Cited By ~ 10

Author(s):

Dominique de SENY ◽

Christelle PROSPERI-MEYS ◽

Carine BEBRONE ◽

Gian Maria ROSSOLINI ◽

Michael I. PAGE ◽

...

Keyword(s):

Bacillus Cereus ◽

Mutational Analysis ◽

Ph Dependence ◽

Catalytic Efficiency ◽

Carbonyl Oxygen ◽

Zinc Ions ◽

Wild Type ◽

Lactam Ring ◽

Hydrolysis Of ◽

Low Activity

The metallo-β-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity, although full efficiency is observed for the di-zinc enzyme. In an attempt to assign the involvement of the different zinc ligands in the catalytic properties of BcII, individual substitutions of selected amino acids were generated. With the exception of His116→Ser (H116S), C221A and C221S, the mono- and di-zinc forms of all the other mutants were poorly active. The activity of H116S decreases by a factor of 10 when compared with the wild type. The catalytic efficiency of C221A and C221S was zinc-dependent. The mono-zinc forms of these mutants exhibited a low activity, whereas the catalytic efficiency of their respective di-zinc forms was comparable with that of the wild type. Surprisingly, the zinc contents of the mutants and the wild-type BcII were similar. These data suggest that the affinity of the β-lactamase for the metal was not affected by the substitution of the ligand. The pH-dependence of the H196S catalytic efficiency indicates that the zinc ions participate in the hydrolysis of the β-lactam ring by acting as a Lewis acid. The zinc ions activate the catalytic water molecule, but also polarize the carbonyl bond of the β-lactam ring and stabilize the development of a negative charge on the carbonyl oxygen of the tetrahedral reaction intermediate. Our studies also demonstrate that Asn233 is not directly involved in the interaction with the substrates.

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