Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection

Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. Th...

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Bibliographic Details
Published in:The FEBS journal 2005-06, Vol.272 (12), p.3085-3092
Main Authors: Castro, Lucila I., Hermsen, Corinna, Schultz, Joachim E., Linder, Jürgen U.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Adenylyl cyclase
Adenylyl Cyclases - genetics
Adenylyl Cyclases - metabolism
Amino Acid Sequence
Asparagine - genetics
Asparagine - metabolism
Bacteria
Catalysis
Catalytic Domain
cyclic nucleotide
Enzymes
Glutamine - genetics
Glutamine - metabolism
Guanosine Triphosphate - metabolism
guanylyl cyclase
Isoenzymes - genetics
Isoenzymes - metabolism
Molecular Sequence Data
Mutation
Mycobacterium tuberculosis
Mycobacterium tuberculosis - enzymology
Substrate Specificity
Tuberculosis
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Summary:Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA‐ATPase and a helix‐turn‐helix DNA‐binding domain. In Rv0386, the standard substrate, adenine‐defining lysine‐aspartate couple is replaced by glutamine‐asparagine. The recombinant adenylyl cyclase domain was active with a Vmax of 8 nmol cAMP·mg−1·min−1. Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side‐activity with GTP as a substrate. Mutation of the glutamine‐asparagine pair either to alanine residues or to the canonical lysine‐aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two noncanonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.
ISSN:1742-464X
1742-4658
DOI:10.1111/j.1742-4658.2005.04722.x