Structural adaptation to selective pressure for altered ligand specificity in the Pseudomonas aeruginosa amide receptor, amiC

The AmiC protein in Pseudomonas aeruginosa is the negative regulator and ligand receptor for an amide-inducible aliphatic amidase operon. In the wild-type PAC1 strain, amidase expression is induced by acetamide or lactamide, but not by butyramide. A mutant strain of P. aeruginosa, PAC181, was selected for its sensitivity to induction by butyramide. The molecular basis for the butyramide inducible phenotype of P.aeruginosa PAC181 has now been determined, and results from a Thr-->Asn mutation at position 106 in PAC181-AmiC. In the wild-type PAC1-AmiC protein this residue forms part of the side wall of the amide-binding pocket but does not interact with the acetamide ligand directly. In the crystal structure of PAC181-AmiC complexed with butyramide, the Thr-->Asn mutation increases the size of the ligand binding site such that the mutant protein is able to close into its 'on' configuration even in the presence of butyramide. Although the mutation allows butyramide to be recognized as an inducer of amidase expression, the mutation is structurally sub-optimal, and produces a significant decrease in the stability of the mutant protein.     

Molecular characterization of AmiC,a positive regulator in acetamidase operon of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterium smegmatis, a rapidly growing non-pathogenic mycobacterium, is currently used as a model organism to study mycobacterial genetics. Acetamidase of M. smegmatis is the highly inducible enzyme of Mycobacteria, which utilizes several amide compounds as sole carbon and nitrogen sources. The acetamidase operon has a complex regulatory mechanism, which involves three regulatory proteins, four promoters, and three operator elements. In our previous study, we showed that over-expression of AmiA leads to a negative regulation of acetamidase by blocking the P2 promoter. In this study, we have identified a new positive regulatory protein, AmiC that interacts with AmiA through protein-protein interaction. Gel mobility shift assay showed that AmiC protein inhibits AmiA from binding to the P2 promoter. Interaction of AmiC with cis-acting elements identified its binding ability to multiple regulatory regions of the operon such as P3, OP3, and P1 promoter/operator. Consequently, the addition of inducer acetamide to AmiC complexe trips the complexes, causing AmiC to appear to be the sensory protein for the amides. Homology modeling and molecular docking studies suggest AmiC as a member of Periplasmic binding proteins, which preferentially bind to the inducers and not to the suppressor. Over-expression of AmiC leads to down-regulation of the negative regulator, amiA, and constitutive up-regulation of acetamidase. Based on these findings, we conclude that AmiC positively regulates the acetamidase operon.     

The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain,a new peptidoglycan binding domain

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram‐negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N‐acetylmuramyl‐L‐alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a β‐sandwich of two symmetrical four‐stranded β‐sheets exposing highly conserved motifs on the two outer faces. We show that this N‐terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan‐binding domain. The C‐terminal catalytic domain shows an auto‐inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity.     

Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process

Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G-protein-coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7-transmembrane domains required for G-protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine-binding protein (LIVBP) and leucine-binding protein (LBP) as already known, and acetamide-binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT-I (1-aminocyclopentane 1,3,4-tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT-I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT-I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.     

A Fail-Safe Mechanism in the Septal Ring Assembly Pathway Generated by the Sequential Recruitment of Cell Separation Amidases and Their Activators

During cytokinesis in Escherichia coli, the peptidoglycan (PG) layer produced by the divisome must be split to promote cell separation. Septal PG splitting is mediated by the amidases: AmiA, AmiB, and AmiC. To efficiently hydrolyze PG, the amidases must be activated by LytM domain factors. EnvC specifically activates AmiA and AmiB, while NlpD specifically activates AmiC. Here, we used an exportable, superfolding variant of green fluorescent protein (GFP) to demonstrate that AmiB, like its paralog AmiC, is recruited to the division site by an N-terminal targeting domain. The results of colocalization experiments indicate that EnvC is recruited to the division site well before its cognate amidase AmiB. Moreover, we show that EnvC and AmiB have differential FtsN requirements for their localization. EnvC accumulates at division sites independently of this essential division protein, whereas AmiB localization is FtsN dependent. Interestingly, we also report that AmiB and EnvC are recruited to division sites independently of one another. The same is also true for AmiC and NlpD. However, unlike EnvC, we find that NlpD shares an FtsN-dependent localization with its cognate amidase. Importantly, when septal PG synthesis is blocked by cephalexin, both EnvC and NlpD are recruited to septal rings, whereas the amidases fail to localize. Our results thus suggest that the order in which cell separation amidases and their activators localize to the septal ring relative to other components serves as a fail-safe mechanism to ensure that septal PG synthesis precedes the expected burst of PG hydrolysis at the division site, accompanied by amidase recruitment.     

Crystallization of and preliminary X-ray data for the negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa.

The negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa has been purified from an over-expressing clone and crystalized. Crystals of diffraction quality were obtained from polyethylene glycol 4000 and ammonium sulphate. AmiC crystallizes in P4(2)2(1)2 (a = 104.4 A, c = 66.6 A) with one subunit in the asymmetric unit. Crystals diffract beyond 2.8 A.     

Inhibition of the aliphatic amidase from Pseudomonas aeruginosa by urea and related compounds.

The time-dependent inhibition of amidase from Pseudomonas aeruginosa strain AI 3 by urea, hydroxyurea and cyanate displayed saturation kinetics fitting a model for the reaction sequence in which formation of a complex in a reversible step was followed by an irreversible step. Altered amidases from mutant strains AIU 1N and OUCH 4, selected for their resistance to inhibition of growth by urea and hydroxyurea respectively, had altered kinetic constants for inhibition indicating reduced binding capacity for the inhibitors. The substrate acetamide protected AI 3 amidase against inhibition by urea,.and altered Ki values for inhibition of the mutant amidases were paralleled by alterations in Km values for acetamide indicating that urea acted at the active site. Inhibition of AI 3 amidase involved the binding of one molecule of urea per molecule of enzyme. Urea inhibited amidase slowly regained activity at pH 7.2 through release of urea.     

Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

Daughter cell separation is controlled by cytokinetic ring‐activated cell wall hydrolysis

During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC^? defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring.     

Expression and purification of a recombinant enantioselective amidase

Microbacterium sp. AJ115 metabolises a wide range of nitriles using the two-step nitrile hydratase/amidase pathway. In this study, the amidase gene of Microbacterium sp. AJ115 has been inserted into the pCal-n-EK expression vector and expressed in Escherichia coli BL21(DE3)pLysS. The expressed protein is active in E. coli and expression of the amidase gene allows E. coli to grow on acetamide as sole carbon and/or nitrogen source. Expression of active amidase in E. coli was temperature dependent with high activity found when cultures were grown between 20 and 30 degrees C but no activity at 37 degrees C. On induction, the amidase represents 28% of the total soluble protein in E. coli. The expressed amidase has been purified in a single step from the crude lysate using the calmodulin-binding peptide (CBP) affinity tag. The V(max) and K(m) of the purified enzyme with acetamide (50 mM) were 4.4 micromol/min/mg protein and 4.5mM, respectively. The temperature optimum was found to be 50 degrees C. Purified enzyme demonstrated enantioselectivity with the ability to preferentially act on the S enantiomer of racemic (R,S)-2-phenylpropionamide. S-2-phenylpropionic acid is produced with an enantiomeric excess of >82% at 50% conversion of the parent amide.     

The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily

The major facilitator superfamily represents the largest group of secondary active membrane transporters in the cell. The 3.3A resolution structure of a member of this protein superfamily, the glycerol-3-phosphate transporter from the Escherichia coli inner membrane, reveals two domains connected by a long central loop. These N- and C-terminal domains, each containing a six-helix bundle, are related by pseudo-twofold symmetry. A substrate translocation pore is located between the two domains and is open to the cytoplasm. Two arginines at the closed end of the pore comprise the substrate-binding site. Biochemical experiments show that, upon substrate binding, the protein adopts a more compact conformation. The crystal structure suggests that the transporter operates through a single binding site, alternating access mechanism via a rocker-switch type of movement of the N- and C-terminal domains. The structure and mechanism of the glycerol-3-phosphate transporter form a paradigm for other members of the major facilitator superfamily.     

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Summary Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI 3. AI 3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2 Compared with AI 3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.     

Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.