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

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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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

Biological Procedures Online ◽

10.1186/s12575-020-00138-0 ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Proteolytic Activity ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00848-16 ◽

2016 ◽

Vol 82 (13) ◽

pp. 3940-3946 ◽

Cited By ~ 8

Author(s):

Seung-Hye Hong ◽

Ho-Phuong-Thuy Ngo ◽

Hyun-Koo Nam ◽

Kyoung-Rok Kim ◽

Lin-Woo Kang ◽

...

Keyword(s):

Retinoic Acid ◽

Bacillus Cereus ◽

Aldehyde Dehydrogenase ◽

Catalytic Efficiency ◽

No Oxidation ◽

Wild Type ◽

Biotechnological Production ◽

Oxidation Activity ◽

Content Type ◽

Bacterial Enzyme

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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Post-VX exposure treatment of rats with engineered phosphotriesterases

Archives of Toxicology ◽

10.1007/s00204-021-03199-6 ◽

2021 ◽

Author(s):

Lisa Stigler ◽

Anja Köhler ◽

Marianne Koller ◽

Laura Job ◽

Benjamin Escher ◽

...

Keyword(s):

Catalytic Efficiency ◽

Wild Type ◽

Single Chain ◽

Major Health ◽

Standard Medical Therapy ◽

Muscarinic Receptor Antagonists ◽

First Time ◽

Hydrolysis Of ◽

Observation Period

AbstractThe biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M−1 min−1) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg−1 i.v.: PTE-2 dosed at 1.3 mg kg−1 i.v. (PTE-2.1) and 2.6 mg kg−1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg−1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

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A variety of roles for versatile zinc in metallo-β-lactamases

Metallomics ◽

10.1039/c4mt00066h ◽

2014 ◽

Vol 6 (7) ◽

pp. 1181-1197 ◽

Cited By ~ 44

Author(s):

A. I. Karsisiotis ◽

C. F. Damblon ◽

G. C. K. Roberts

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Zinc Ions ◽

Lactam Ring ◽

Hydrolysis Of

β-Lactamases inactivate the important β-lactam antibiotics by catalysing the hydrolysis of the β-lactam ring, thus. One class of these enzymes, the metallo-β-lactamases, bind two zinc ions at the active site and these play important roles in the catalytic mechanism.

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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

10.21203/rs.3.rs-62284/v1 ◽

2020 ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Km Value ◽

Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.

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Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

Journal of Bacteriology ◽

10.1128/jb.00259-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6179-6183 ◽

Cited By ~ 16

Author(s):

Jung-Kul Lee ◽

Ee-Lui Ang ◽

Huimin Zhao

Keyword(s):

Substrate Specificity ◽

Mutational Analysis ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Saturation Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Enzyme Substrate ◽

Molecular Determinants

ABSTRACT Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

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Mutational Analysis of the Yeast DEAH-Box Splicing Factor Prp16

Genetics ◽

10.1093/genetics/149.2.807 ◽

1998 ◽

Vol 149 (2) ◽

pp. 807-815 ◽

Cited By ~ 1

Author(s):

Hans-Rudolf Hotz ◽

Beate Schwer

Keyword(s):

Amino Acids ◽

Mutational Analysis ◽

Splicing Factor ◽

Splicing Factors ◽

Sequence Motifs ◽

Wild Type ◽

Alanine Scanning Mutagenesis ◽

Alanine Scan ◽

Hydrolysis Of

Abstract Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates. Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 were tested for in vivo function by complementation of a Δprp16 null strain. In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373–377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable. The S505A and T507A mutants in motif III (SAT) were viable. In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth. Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan. Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells. Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo. These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors.

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Mutational Analysis of a Novel Mutation (p.H232R) in the SRD5A2 Causing 46,XY Disorder of Sex Development

10.21203/rs.3.rs-786500/v1 ◽

2021 ◽

Author(s):

Liwei Li ◽

Junhong Zhang ◽

Qing Li ◽

Li Qiao ◽

Pengcheng Li ◽

...

Keyword(s):

Molecular Mechanism ◽

Mutational Analysis ◽

Novel Mutation ◽

Catalytic Efficiency ◽

Hek293 Cells ◽

Compound Heterozygous ◽

Wild Type ◽

Disorder Of Sex Development ◽

Maternal Grandfather ◽

Sex Development

Abstract Background: Over 100 mutations in SRD5A2 gene have been identified in subjects with 46,XY DSD. Exploring SRD5A2 mutation and elucidating its molecular mechanism will find the domains function of 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.H232R/p.Q6X mutation of SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in SRD5A2 gene causes 46,XY DSD occurrence is needed to be further explored. Results: In order to clarify the cause of 46,XY DSD in the case’s family, SRD5A2 sequencing were performed. The heterozygous p.H232R mutation were identified in the case’s father, so we concluded that the heterozygous p.H232R mutation originated from paternal family and didn’t cause 46,XY DSD occurrence. The heterozygous p.Q6X mutation were identified in the case’s mother, maternal uncle and maternal grandfather, indicating that the heterozygous p.Q6X mutation descended from maternal family and didn’t cause 46,XY DSD occurrence. In order to clarify p.H232R mutation in SRD5A2 on DHT production, p.H232R mutant SRD5A2 plasmids were transfected with HEK293 cells and LC-MS detected that DHT production decreased compared with wild-type SRD5A2 infected ones.Conclusions: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuated 5α-reductase 2 enzymatic catalytic efficiency.

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Mutational Analysis of nocK and nocL in the Nocardicin A Producer Nocardia uniformis

Journal of Bacteriology ◽

10.1128/jb.187.2.739-746.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 739-746 ◽

Cited By ~ 17

Author(s):

Wendy L. Kelly ◽

Craig A. Townsend

Keyword(s):

Natural Products ◽

Gene Cluster ◽

Mutational Analysis ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Wild Type ◽

Nocardicin A ◽

Lactam Ring ◽

Undetermined Function

ABSTRACT The nocardicins are a family of monocyclic β-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp. tsuyamanensis ATCC 21806. The most potent of this series is nocardicin A, containing a syn-configured oxime moiety, an uncommon feature in natural products. The nocardicin A biosynthetic gene cluster was recently identified and found to encode proteins in keeping with nocardicin A production, including the nocardicin N-oxygenase, NocL, in addition to genes of undetermined function, such as nocK, which bears similarities to a broad family of esterases. The latter was hypothesized to be involved in the formation of the critical β-lactam ring. While previously shown to effect oxidation of the 2′-amine of nocardicin C to provide nocardicin A, it was uncertain whether NocL was the only N-oxidizing enzyme required for nocardicin A biosynthesis. To further detail the role of NocL in nocardicin production in N. uniformis, and to examine the function of nocK, a method for the transformation of N. uniformis protoplasts to inactivate both nocK and nocL was developed and applied. A reliable protocol is reported to achieve both insertional disruption and in trans complementation in this strain. While the nocK mutant still produced nocardicin A at levels near that seen for wild-type N. uniformis, and therefore has no obvious role in nocardicin biosynthesis, the nocL disruptant failed to generate the oxime-containing metabolite. Nocardicin A production was restored in the nocL mutant upon in trans expression of the gene. Furthermore, the nocL mutant accumulated the biosynthetic intermediate nocardicin C, confirming its role as the sole oxime-forming enzyme required for production of nocardicin A.

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The pH-dependence and group modification of β-lactamase I

Biochemical Journal ◽

10.1042/bj1490547 ◽

1975 ◽

Vol 149 (3) ◽

pp. 547-551 ◽

Cited By ~ 27

Author(s):

S G Waley

Keyword(s):

Ph Dependence ◽

Water Soluble ◽

Side Chain ◽

Polar Group ◽

Woodward’S Reagent K ◽

Group Modification ◽

Penicillanic Acid ◽

Lactam Ring ◽

Parameter Versus ◽

Hydrolysis Of

The pH-dependence of the kinetic parameters for the hydrolysis of the β-lactam ring by β-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-α-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of β-lactamase I.

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