The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (Km) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants kcat and Km revealed a change in the pKa of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pKa values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base

Co-Catalytic Metallopeptidases as Pharmaceutical Targets

Understanding the reaction mechanism of co-catalytic metallopeptidases provides a starting point for the design and synthesis of new molecules that can be screened as potential pharmaceuticals. Many of the enzymes that contain co-catalytic metallo-active sites play important roles in cellular processes such as tissue repair, protein maturation, hormone level regulation, cell-cycle control and protein degradation. Therefore, these enzymes play central roles in several disease states including cancer, HIV, stroke, diabetes, bacterial infections, neurological processes, schizophrenia, seizure disorders, and amyotrophic lateral sclerosis. The mechanism of AAP, an aminopeptidase from Aeromonas proteolytica, is one of the best-characterized examples of a metallopeptidase containing a co-catalytic metallo-active site, although this enzyme is not a specific pharmaceutical target at this time. As a large majority of co-catalytic metallopeptidases contain active sites that are nearly identical to the one observed in AAP, the major steps of their catalytic mechanisms are likely to be very similar. With this in mind, it is possible to propose a general catalytic mechanism for the hydrolysis of amino acid substrates

The High-Resolution Structures of the Neutral and the Low pH Crystals of Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-Å resolution at neutral pH and at 1.24-Å resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases

Function of the Signal Peptide and N- and C-terminal Propeptides in the Leucine Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP\u27s native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki∼0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis

Spectroscopic and Thermodynamic Characterization of the E151D and E151A Altered Leucine Aminopeptidases from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 Å in carboxypeptidase G2 and 3.30 and 3.63 Å in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV−vis and electron paramagnetic resonance (EPR) spectroscopy. UV−vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV−vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M-1 cm-1 vs. 56 and 109 M-1 cm-1 for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g⊥ = 2.35, g∥ = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) gx,y = 2.509, gz = 2.19, E/D = 0.19, A = 0.0069 cm-1 and (ii) gx,y = 2.565, gz = 2.19, E/D = 0.20, A = 0.0082 cm-1 indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with Kd values elevated by factors of ∼850 and ∼24 000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP

Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the d-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min-1, which is over 2000 times slower than r AAP (4380 min-1). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV−vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and Km for the hydrolysis of l-leucine p-nitroanilide (l-pNA) revealed a change in an ionization constant in the enzyme−substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 Å resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate

Two Translation Products of <i>Yersinia yscQ</i> Assemble To Form a Complex Essential to Type III Secretion

The bacterial flagellar C-ring is composed of two essential proteins, FliM and FliN. The smaller protein, FliN, is similar to the C-terminus of the larger protein, FliM, both being composed of SpoA domains. While bacterial type III secretion (T3S) systems encode many proteins in common with the flagellum, they mostly have a single protein in place of FliM and FliN. This protein resembles FliM at its N-terminus and is as large as FliM but is more like FliN at its C-terminal SpoA domain. We have discovered that a FliN-sized cognate indeed exists in the <i>Yersinia</i> T3S system to accompany the FliM-sized cognate. The FliN-sized cognate, YscQ-C, is the product of an internal translation initiation site within the locus encoding the FliM-sized cognate YscQ. Both intact YscQ and YscQ-C were found to be required for T3S, indicating that the internal translation initiation site, which is conserved in some but not all YscQ orthologs, is crucial for function. The crystal structure of YscQ-C revealed a SpoA domain that forms a highly intertwined, domain-swapped homodimer, similar to those observed in FliN and the YscQ ortholog HrcQ_B. A single YscQ-C homodimer associated reversibly with a single molecule of intact YscQ, indicating conformational differences between the SpoA domains of intact YscQ and YscQ-C. A “snap-back” mechanism suggested by the structure can account for this. The 1:2 YscQ–YscQ-C complex is a close mimic of the 1:4 FliM–FliN complex and the likely building block of the putative <i>Yersinia</i> T3S system C-ring

Mycothiol Import by Mycobacterium smegmatis and Function as a Resource for Metabolic Precursors and Energy Production▿

Mycothiol ([MSH] AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn1) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-14C]cysteine or with [6-3H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo-inositol can serve as a sole carbon source for growth of M. smegmatis, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ∼50 h in strains with active MshC and 16 ± 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis

The 1.20 Å Resolution Crystal Structure of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e Complexed with Tris: A Tale of Buffer Inhibition

The aminopeptidase from Aeromonas proteolytica (AAP) is a bridged bimetallic enzyme that removes the N-terminal amino acid from a peptide chain. To fully understand the metal roles in the reaction pathway of AAP we have solved the 1.20 Å resolution crystal structure of native AAP (PDB ID = 1LOK). The high-quality electron density maps showed a single Tris molecule chelated to the active site Zn2+, alternate side chain conformations for some side chains, a sodium ion that mediates a crystal contact, a surface thiocyanate ion, and several potential hydrogen atoms. In addition, the high precision of the atomic positions has led to insight into the protonation states of some of the active site amino acid side chains

Observation of an E2 (Ubc9)-homodimer by crystallography

Post-translational modifications by the small ubiquitin-like modifiers (SUMO), in particular the formation of poly-SUMO-2 and -3 chains, regulates essential cellular functions and its aberration leads to life-threatening diseases (Geoffroy and Hay, 2009) [1]. It was shown previously that the non-covalent interaction between SUMO and the conjugating enzyme (E2) for SUMO, known as Ubc9, is required for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. However, the structure of SUMO-Ubc9 non-covalent complex, by itself, could not explain how the poly-SUMO-2/3 chain forms and consequently a Ubc9 homodimer, although never been observed, was proposed for poly-SUMO-2/3 chain formation (Knipscheer et al., 2007) [2]. Here, we solved the crystal structure of a heterotrimer containing a homodimer of Ubc9 and the RWD domain from RWDD3. The asymmetric Ubc9 homodimer is mediated by the N-terminal region of one Ubc9 molecule and a surface near the catalytic Cys of the second Ubc9 molecule (Fig. 1A). This N-terminal surface of Ubc9 that is involved in the homodimer formation also interacts with the RWD domain, the ubiquitin-fold domain of the SUMO activating enzyme (E1), SUMO, and the E3 ligase, RanBP2 (Knipscheer et al., 2007; Tong et al.. 1997; Tatham et al., 2005; Reverter and Lima, 2005; Capili and Lima, 2007; Wang et al., 2009, 2010; Wang and Chen, 2010; Alontaga et al., 2015) [2–10]. The existence of the Ubc9 homodimer in solution is supported by previously published solution NMR studies of rotational correlation time and chemical shift perturbation (Alontaga et al., 2015; Yuan et al., 1999) [10,11]. Site-directed mutagenesis and biochemical analysis suggests that this dimeric arrangement of Ubc9 is likely important for poly-SUMO chain formation (Fig. 1B and C). The asymmetric Ubc9 homodimer described for the first time in this work could provide the critical missing link in the poly-SUMO chain formation mechanism. The data presented here are related to the research article entitled, “RWD domain as an E2 (Ubc9) interaction module” (Alontaga et al., 2015) [10]. The data of the crystal structure has been deposited to RCSB protein data bank with identifier: 4Y1L. Keywords: SUMO, Poly-SUMO chain, RWD, Ubiquitin, Ubc9, E1, Ubiquitin-like, Modification