Structural and kinetic studies on adenylosuccinate lyase from Mycobacterium smegmatis and Mycobacterium tuberculosis provide new insights on the catalytic residues of the enzyme

• Published in: The FEBS journal 2014-03, Vol.281 (6), p.1642-1658
• Main Authors: Banerjee, Sanchari, Agrawal, Monika J., Mishra, Diptimayee, Sharan, Siddharth, Balaram, Hemalatha, Savithri, Handanhal S., Murthy, Mathur R. N.
• Format: Article
• Language: English
• Subjects: adenylosuccinate lyase; Adenylosuccinate Lyase - chemistry; Adenylosuccinate Lyase - genetics; Adenylosuccinate Lyase - metabolism; Amino Acid Sequence; Apoenzymes - chemistry; Apoenzymes - genetics; Apoenzymes - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biosynthesis; catalytic activity; Catalytic Domain - genetics; Crystallography, X-Ray; Enzymes; Kinetics; Models, Molecular; Molecular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - genetics; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Pharmacology; Phylogeny; Protein Conformation; Protein Structure, Quaternary; purine nucleotide supply; Sequence Homology, Amino Acid; slow growth rate; Static Electricity; Structural Homology, Protein; Tuberculosis; X‐ray crystallography
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12730

Adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis, has been recognized as a drug target against microbial infections. In the present study, ASL from Mycobacterium smegmatis (MsASL) and Mycobacterium tuberculosis (MtbASL) were cloned, purified and crystallized. The X‐ray crystal structure of MsASL was determined at a resolution of 2.16 Å. It is the first report of an apo‐ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. These structures suggest that His149 and either Lys285 or Ser279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. Most of the active site residues were found to be conserved, with the exception of Ser148 and Gly319 of MsASL. Ser148 is structurally equivalent to a threonine in most other ASLs. Gly319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria and their high GC containing genomes, as well as their dependence on other salvage pathways for the supply of purine nucleotides. Structured digital purB and purB bind by x-ray crystallography (View interaction) X‐ray crystal structure of Mycobacterium smegmatis apo‐adenylosuccinate lyase (ASL) with a partially ordered active site loop was determined at 2.16 Å resolution. Comparative analysis of ASL structures suggests that His149, Lys285 and Ser279 are likely to be functionally important. The low catalytic activity observed for M. smegmatis and M. tuberculosis ASLs is consistent with their slow growth rate.