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Applied and Environmental Microbiology, January 2009, p. 212-223, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01322-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Analysis of MmeI from Methanol Utilizer Methylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes{triangledown}

Joanna Nakonieczna,1,2* Tadeusz Kaczorowski,2* Agnieszka Obarska-Kosinska,3,4 and Janusz M. Bujnicki3,5

Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland,1 Department of Microbiology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland,2 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland,3 Institute of Biochemistry and Biophysics PAS, Pawinskiego 5A, 02-106 Warsaw, Poland,4 Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 98, 61-614 Poznan, Poland5

Received 13 June 2008/ Accepted 29 October 2008

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-L-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N6-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D70-X9-EXK82, characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N6-adenine {gamma}-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

* Corresponding author. Mailing address: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk, and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. Phone for Joanna Nakonieczna: 48 58 523 63 22. Fax: 48 58 523 64 26. E-mail: strzala{at}biotech.ug.gda.pl. Phone for Tadeusz Kaczorowski: 48 58 305 62 42. Fax: 48 58 523 64 20. E-mail: kaczorow{at}biotech.ug.gda.pl

{triangledown} Published ahead of print on 7 November 2008.

Applied and Environmental Microbiology, January 2009, p. 212-223, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01322-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Morgan, R. D., Luyten, Y. A. (2009). Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity. Nucleic Acids Res 37: 5222-5233 [Abstract] [Full Text]  
  • Morgan, R. D., Dwinell, E. A., Bhatia, T. K., Lang, E. M., Luyten, Y. A. (2009). The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection. Nucleic Acids Res 37: 5208-5221 [Abstract] [Full Text]  


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