Arginase is a bimetallic enzyme that utilizes mainly Mn 2+ or Co 2+ for catalytic function. In human homolog, substitution of Mn 2+ with Co 2+ significantly reduces the K m value without affecting the k cat . However, in the Helicobacter pylori counterpart (important for pathogenesis), the k cat increases nearly 4-fold with Co 2+ ions both in the recombinant holoenzyme and arginase isolated from H. pylori grown with Co 2+ or Mn 2+ . This suggests that the active site of arginase in the two homologs is modulated differently by these two metal ions. To investigate the underlying mechanism for metal-induced difference in catalytic activity in the H. pylori enzyme, we used biochemical, biophysical and microsecond MD simulations studies. The study shows that the difference in binding affinity of Co 2+ and Mn 2+ ions with the protein is linked to a different positioning of a loop (-- 122 HTAYDSDSKHIHG 134 --) that contains a conserved catalytic His133. Consequently, the proximity of His133 and conserved Glu281 is varied. We found that the Glu281-His133 interaction is crucial for catalytic function and was previously unexplored in other homologs. We suggest that the proximity difference between these two residues in the Co 2+ - and Mn 2+ -proteins alters the proportion of protonated His133 via variation in its p K a . This affects the efficiency of proton transfer-an essential step of L-arginine hydrolysis reaction catalyzed by arginase and thus activity. Unlike in human arginase, the flexibility of the above segment observed in H. pylori homolog suggests that this region in the H. pylori enzyme may be explored to design its specific inhibitors.
The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis ( Mt DnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of Mt DnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis , a complex was modeled using the Mt DnaG-CTD and Mt DnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of Mt DnaG were identified as potential key residues interacting with Mt DnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of Mt DnaB-NTD with the Ile605Ala mutant of Mt DnaG-CTD compared with native Mt DnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, Mt DnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae . The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.
Cysteine biosynthesis takes place via a two-step pathway in bacteria, fungi, plants and protozoan parasites, but not in humans, and hence, the machinery of cysteine biosynthesis is an opportune target for therapeutics. The decameric cysteine synthase complex (CSC) is formed when the C-terminal tail of serine acetyltransferase (SAT) binds in the active site of O -acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS from Brucella abortus (BaOASS) does not interact with its cognate SAT C-terminal tail. Crystal structures of native BaOASS showed that residues Gln96 and Tyr125 occupy the active-site pocket and interfere with the entry of the SAT C-terminal tail. The BaOASS (Q96A–Y125A) mutant showed relatively strong binding ( K d = 32.4 μM) to BaSAT C-terminal peptides in comparison with native BaOASS. The mutant structure looks similar except that the active-site pocket has enough space to bind the SAT C-terminal end. Surface plasmon resonance results showed a relatively strong (7.3 μM K d ) interaction between BaSAT and the BaOASS (Q96A–Y125A), but no interaction with native BaOASS. Taken together, our observations suggest that the CSC does not form in B. abortus .