TY - JOUR
T1 - Isolation and biochemical characterization of the PilA protein of Neisseria meningitidis
AU - Arvidson, Cindy Grove
AU - So, Magdalene
N1 - Funding Information:
1The DNA sequence used has been deposited in GenBank under Accession No. AF003940. 2This work was supported by NIH Grant R01 AI34560 to M.S. 3To whom correspondence should be addressed. Fax: 503-494-6862. E-mail: arvidson@ohsu.edu.
PY - 1997/12/15
Y1 - 1997/12/15
N2 - PilA is the response regulator of a two-component regulatory system that controls a number of genes in the pathogenic Neisseria. Previous work has shown that Neisseria gonorrhoeae (GC) PilA binds DNA and also hydrolyzes GTP. Here, we report the cloning, sequencing, purification, and biochemical characterization of PilA from N. meningitidis (MC) strain 8013, MC pilA is 94% identical to GC pilA at the nucleotide level. Of the 78 nucleotide changes, 52 are silent, while 26 result in a total of 20 amino acid changes. Additionally, the MC homolog has a 4-amino-acid insertion between the putative DNA-binding and GTP-binding domains. Purified MC PilA binds to the same DNA fragment we have previously shown to be bound by GC PilA specifically and also hydrolyzes GTP. The K(m) of MC PilA for GTP is 8.6 μM similar to that determined for the GC protein. However, the maximum velocity (V(max)) is ~35-fold greater than the GC PilA activity. Additionally, the nucleotide specificity of MC PilA differs from that of GC PilA. While GC PilA hydrolyzes only GTP, MC PilA hydrolyzes GTP and ATP equally well, and CTP and UTP also compete for this activity.
AB - PilA is the response regulator of a two-component regulatory system that controls a number of genes in the pathogenic Neisseria. Previous work has shown that Neisseria gonorrhoeae (GC) PilA binds DNA and also hydrolyzes GTP. Here, we report the cloning, sequencing, purification, and biochemical characterization of PilA from N. meningitidis (MC) strain 8013, MC pilA is 94% identical to GC pilA at the nucleotide level. Of the 78 nucleotide changes, 52 are silent, while 26 result in a total of 20 amino acid changes. Additionally, the MC homolog has a 4-amino-acid insertion between the putative DNA-binding and GTP-binding domains. Purified MC PilA binds to the same DNA fragment we have previously shown to be bound by GC PilA specifically and also hydrolyzes GTP. The K(m) of MC PilA for GTP is 8.6 μM similar to that determined for the GC protein. However, the maximum velocity (V(max)) is ~35-fold greater than the GC PilA activity. Additionally, the nucleotide specificity of MC PilA differs from that of GC PilA. While GC PilA hydrolyzes only GTP, MC PilA hydrolyzes GTP and ATP equally well, and CTP and UTP also compete for this activity.
KW - ATP hydrolysis
KW - DNA-binding
KW - GTP hydrolysis
KW - Neisseria meningitidis
KW - PilA
KW - Transcriptional regulation
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U2 - 10.1006/abbi.1997.0399
DO - 10.1006/abbi.1997.0399
M3 - Article
C2 - 9434748
AN - SCOPUS:0031574452
SN - 0003-9861
VL - 348
SP - 357
EP - 362
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -