Kinetic and structural characterization of bacterial glutaminyl cyclases from Zymomonas mobilis and Myxococcus xanthus

Abstract

Although enzymes responsible for the cyclization of amino-terminal glutamine residues are present in both plant and mammal species, none have yet been characterized in bacteria. Based on low sequence homologies to plant glutaminyl cyclases (QCs), we cloned the coding sequences of putative microbial QCs from Zymomonas mobilis (ZmQC) and Myxococcus xanthus (MxQC). The two recombinant enzymes exhibited distinct QC activity, with specificity constants k(cat)/K(m) of 1.47±0.33 mm⁻¹ s⁻¹ (ZmQC) and 142±32.7 mm⁻¹ s⁻¹ (MxQC) towards the fluorescent substrate glutamine-7-amino-4-methyl-coumarine. The measured pH-rate profile of the second order rate constant displayed an interesting deviation towards the acidic limb of the pH chart in the case of ZmQC, whereas MxQC showed maximum activity in the mild alkaline pH range. Analysis of the enzyme variants ZmQCGlu⁴⁶Gln and MxQCGln⁴⁶Glu show that the exchanged residues play a significant role in the pH behaviour of the respective enzymes. In addition, we determined the three dimensional crystal structures of both enzymes. The tertiary structure is defined by a five-bladed β-propeller anchored by a core cation. The structures corroborate the putative location of the active site and confirm the proposed relation between bacterial and plant glutaminyl cyclases.

Structure of the host-recognition device of Staphylococcus aureus phage ϕ11

“…The highest score (Z = 16.1, r.m.s.d. = 3.1 Å), however, was obtained for the enzyme glutamine cyclotransferase from Zymomonas mobilis (PDB code 3NOL) 29 . To our knowledge, only two other examples of β-propellers in putative RBPs have been reported.…”

“…When the Zymomonas mobilis glutamine cyclotransferase structure 29 was superimposed onto the RBP propeller domain, the active site of the enzyme overlaid a deep crevice located within the RBP lower face ( Fig. 4 ).…”

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

“…PorC proteoforms were characterized by a mass discrepancy of −19 Da, in agreement with the presence of a disulfide bond (−2 Da) and the cyclization of the N-terminal glutamyl residue on signal peptide cleavage, yielding a pyroglutamyl residue (−17 Da) (Table S2). Although such N-terminal protein modifications (i.e., N-formylation and the formation of pyroglutamyl residues) have been identified in bacterial secreted proteins and OMPs (13,14), we sought additional evidence about the natural relevance of secreted porin species. To check whether nonacylated soluble porins resulted from a physiological secretion process, endogenous proteins secreted from stationary-phase, untransformed C. glutamicum cells were isolated using an identical protocol.…”

Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight

“…Glutamine is particularly susceptible; under physiological conditions it cyclizes to OP at the rate of 10% per day (9). N-terminal glutaminyl and glutamyl residues of polypeptides can likewise spontaneously cyclize to OP residues; this reaction is also catalyzed by glutaminyl-peptide cyclotransferase (12,13). The enzyme pyroglutamyl peptidase, which occurs in all domains of life, removes N-terminal OP residues, yielding free OP (14).…”