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Ribokinase (RK) is an ATP dependent sugar kinase that enables the entry of ribose in the metabolism. Leishmania accumulates ribose into the cytosol through hydrolysis of nucleosides and by transport from the extracellular environment. Activation by RK is critical to mobilize the ribose into the metabolism of Leishmania. To understand the catalytic role, the crystal structure of RK (LdRK) from L. donovani was determined in the apo and complex forms with several nucleotides (ATP, AMPPCP and ADP) in the presence of Na + ion. The dual insertion of five amino acid stretches makes LdRK structurally unique from other reported structures of RKs. The structure of LdRK-ATP provided the basis for positioning of γ-phosphate of ATP by conserved -GAGD- motif. Liganded and unliganded structures of LdRK exists in similar conformation, which suggests binding of nucleotides does not make any significant conformational changes in nucleotide-bound structures. Substitution of a conserved asparagine with phenylalanine in ribose binding pocket differentiates the LdRK from other RKs. Glycerol molecule bound in the substrate binding pocket mimics the enzyme-substrate interactions but in turn, hampers the binding of ribose to LdRK. Comparative structural analysis revealed the flexibility of γ-phosphate, which adopts multiple conformations in the absence of divalent metal ion and ribose. Similar to other RKs, LdRK is also dependent on monovalent as well as divalent cations for its catalytic activity., (Copyright © 2019. Published by Elsevier B.V.)

Methionine aminopeptidases (MetAPs) are a class of enzymes evolved to cleave initiator methionine in 60-70% of the total cellular proteins in all living cells. Based on their sequence differences, they are classified into Type 1 and Type 2. Type 1 is further divided into Type 1a, 1a', 1b, 1c and 1d. Irrespective of various classifications, all MetAPs reported till date displayed hydrolytic activity against peptides that contain only methionine on the N-terminus. A cysteine at the top of the active site in all the Type 1 structures is reported to be critical for the specificity. Mutation of this cysteine to serine or asparagine leads to loss of specificity. In the present study, we have identified a class of MetAPs in some of the proteobacteria that have an asparagine at this site. Most of the proteobacteria that contain MetAP1n are pathogenic in nature. Biochemical and structural studies on two proteins, one from each of V. coralliilyticus and K. pneumoniae confirm that these enzymes cleave leucine in addition to methionine. Crystallographic and homology modeling studies suggest that relaxed substrate specificity of this new class of enzymes could be due to the increased flexibility in the active site. Since this new class has an asparagine at the critical position that probably contributes for the relaxed substrate specificity and also differentiates them from other Type 1 MetAPs, we classified them as Type 1n., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aminopeptidases catalyze the hydrolysis of amino acids from the N-terminus of protein or peptide substrates. M1 family aminopeptidases are important for the pathogenicity of bacteria and play critical role in many physiological processes such as protein maturation, regulation of peptide hormone levels in humans. Most of the M1 family aminopeptidases reported till date display broad substrates specificity, mostly specific to basic and hydrophobic residues. In the current study we report the discovery of a novel M1 class aminopeptidase from Legionella pneumophila (LePepA), which cleaves only acidic residues. Biochemical and structural studies reveal that the S1 pocket is polar and positively charged. Bioinformatic analysis suggests that such active site is unique to only Legionella species and probably evolved for special needs of the microbe. Given its specific activity, LePepA could be useful in specific biotechnological applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)