Modification of the Transfructosylation Activity of Bacillus Subtilis Levansucrase by Solvent Effect and Site-Directed Mutagenesis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

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Mass Spectrometry and Site-directed Mutagenesis Identify Several Autophosphorylated Residues Required for the Activity of PrkC, a Ser/Thr Kinase from Bacillus subtilis

Journal of Molecular Biology ◽

10.1016/s0022-2836(03)00579-5 ◽

2003 ◽

Vol 330 (3) ◽

pp. 459-472 ◽

Cited By ~ 55

Author(s):

Edwige Madec ◽

Allan Stensballe ◽

Sven Kjellström ◽

Lionel Cladière ◽

Michal Obuchowski ◽

...

Keyword(s):

Mass Spectrometry ◽

Bacillus Subtilis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

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Site-directed mutagenesis of active site residues in Bacillus subtilis α-amylase

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(92)90249-d ◽

1992 ◽

Vol 1120 (3) ◽

pp. 281-288 ◽

Cited By ~ 42

Author(s):

Kenji Takase ◽

Takashi Matsumoto ◽

Hiroshi Mizuno ◽

Kazio Yamane

Keyword(s):

Bacillus Subtilis ◽

Active Site ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Active Site Residues

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Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.16.6238 ◽

1990 ◽

Vol 87 (16) ◽

pp. 6238-6242 ◽

Cited By ~ 172

Author(s):

M. J. Weickert ◽

G. H. Chambliss

Keyword(s):

Bacillus Subtilis ◽

Catabolite Repression ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Operator Sequence

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Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

10.21203/rs.3.rs-409769/v1 ◽

2021 ◽

Author(s):

Jing Wu ◽

Wei Li ◽

Shiguang Zhao ◽

Sen-he Qian ◽

Zhou Wang ◽

...

Keyword(s):

Bacillus Subtilis ◽

Electron Transport ◽

Biofilm Formation ◽

Stable State ◽

Transcriptional Regulator ◽

Site Directed Mutagenesis ◽

Expression Level ◽

Transcriptional Analysis ◽

Directed Mutagenesis ◽

Nadh Dehydrogenases

Abstract Background: MK-7 is a highly valuable vitamin K2 produced by Bacillus subtilis. Biofilm forming caused by quorum sensing (QS) system was beneficial for MK-7 synthesis. However, the specific regulator in QS system which play the most significant role in biofilm formation and MK production have not been revealed at genetic level, and this limits the possibility of further increasing MK-7 production.Results: In this study, the influence of different mutants in QS system on biofilm formation and MK-7 production was investigated. The transcriptional regulator SinR was chosen finally because the complete knocking out of sinR (KO-SinR) maximized the biofilm biomass and increased the yield of MK-7. However, KO-SinR led to a large number of spore and wrinkle forming, which slow down even stop the MK-7 biosynthesis. Five SinR mutants (E97K, Y101L, W104K, R105S, SinRquad) were constructed by site-directed mutagenesis of four aromatic residues (Glu97, Tyr101, Trp104 and Arg105). Among these, SinRquad formed more wrinkly but smooth biofilm, and obtained the maximum MK-7 value (102.56±2.84 mg/L), which was ten times of the parent strain. Comparative transcriptional analysis showed that SinRquad promoted the synthesis of extracellular polymeric substances and inhibited the swimming motility of late-flagellar. In addition, SinRquad upregulated the expression level of ctaC-G operator and qcrA-C operator which encode the cytochromes. Upregulation of components in the oxidative respiratory chain of B. subtilis was due to the upregulation of NADH dehydrogenases. Most NADH dehydrogenases especially sdhA-C and glpD, increased 1.01-, 3.93-, 1.87-, 1.11- fold, respectively. The increase in expression level of NADH dehydrogenases indicated the ratio of NADH/NAD+ decreased and more electrons produced for the electron transport system (ETM). Wrinkly and smooth biofilm formed a network of interconnected channels with low resistance to liquid flow was beneficial to obtain a more stable state of electron transport chain, which facilitate the metabolic flux of four modules of MK-7 synthesis pathways.Conclusions: In this study, we report for the first time that SinRquad has significant effects on the MK-7 synthesis by forming wrinkly and smooth biofilm, upregulating the expression level of most NADH dehydrogenases, providing more stable state of the electron transport.

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Characterization of the Interaction of Bacillus subtilis PyrR with pyr mRNA by Site-Directed Mutagenesis of the Protein

Journal of Bacteriology ◽

10.1128/jb.184.9.2521-2528.2002 ◽

2002 ◽

Vol 184 (9) ◽

pp. 2521-2528 ◽

Cited By ~ 13

Author(s):

Heather K. Savacool ◽

Robert L. Switzer

Keyword(s):

Bacillus Subtilis ◽

Rna Binding ◽

Site Directed Mutagenesis ◽

Crystallographic Structure ◽

Directed Mutagenesis ◽

Dependent Manner ◽

Missense Mutations ◽

Uracil Phosphoribosyltransferase ◽

Pyrimidine Nucleotide ◽

Uridine Nucleotide

ABSTRACT The Bacillus subtilis PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (pyr) operon by binding in a uridine nucleotide-dependent manner to specific sites on pyr mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription. The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA. Missense mutations were constructed and evaluated for their effects on regulation of pyr genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR. A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind pyr RNA in vitro. On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding. Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly. Arg-19 was implicated in pyr regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form. The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure. The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species.

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