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• W2078833893 abstract "Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an α-subunit, a spacer sequence, and a β-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with α- and β-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1β onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1β to generate a strong nucleophile and to direct the OG atom of the Ser-1β to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme. Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an α-subunit, a spacer sequence, and a β-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with α- and β-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1β onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1β to generate a strong nucleophile and to direct the OG atom of the Ser-1β to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme. Cephalosporin acylase (CA) 1The abbreviations used are: CA, cephalosporin acylase; Ntn, N-terminal; PS, proteasome β subunit; GA, glycosyl asparaginase; CAD, a class I CA from P. diminuta KAC-1; r.m.s., root mean square. is a new family of the N-terminal (Ntn) hydrolase superfamily that is defined by the SCOP data base (1.Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5606) Google Scholar). The Ntn hydrolase superfamily is defined by SCOP as a motif containing four layers of α-helices and β-sheets in a αββα fashion. There are five known families of the Ntn hydrolase superfamily; they are the class II glutamine amidotransferases, penicillin G acylase, penicillin V acylase, proteasome β subunit, and glycosyl asparaginase (1.Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5606) Google Scholar). Ntn hydrolases are generally translated into a precursor enzyme. Several Ntn hydrolases are activated through auto-proteolytic processing by the Ser, Thr, or Cys residues. Auto-proteolytic processing occurs in an intramolecular manner in several Ntn hydrolases. These include glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase, penicillin G acylase, proteasome β subunit (PS), glycosyl asparaginase (GA), and cephalosporin acylase (2.Murzin A.G. Curr. Opin. Struct. Biol. 1996; 6: 386-394Crossref PubMed Scopus (224) Google Scholar, 3.Lee Y.S. Park S.S. J. Bacteriol. 1998; 180: 4576-4582Crossref PubMed Google Scholar, 4.Ditzel L. Huber R. Mann K. Heinemeyer W. Wolf D.H. Groll M. J. Mol. Biol. 1998; 279: 1187-1191Crossref PubMed Scopus (105) Google Scholar). The gene structure of the open reading frame of CAs generally consists of a signal peptide that is followed by an α-subunit, a spacer sequence, and a β-subunit, all of which are translated into a single polypeptide chain, the CA precursor. The precursor is post-translationally modified into an active heterodimeric enzyme with α and β subunits, first by intramolecular cleavage process and, second, by intermolecular cleavage process. The heterodimeric enzyme is envisaged from the structural studies of the active CA and precursor CA, in which the nascent polypeptide (precursor) of cephalosporin acylase is autoproteolytically activated through a two-step autocatalytic process upon folding (3.Lee Y.S. Park S.S. J. Bacteriol. 1998; 180: 4576-4582Crossref PubMed Google Scholar, 5.Li Y. Chen J. Jiang W. Mao X. Zhao G. Wang E. Eur. J. Biochem. 1999; 262: 713-719Crossref PubMed Scopus (68) Google Scholar). The first step is an intramolecular cleavage process by the N-terminal Ser at the beginning of the β-subunit. This results in an α-subunit, a spacer peptide that is attached to the C terminus of the α-subunit, and a β-subunit. The second step is an intermolecular event, which is mediated by the newly generated N-terminal Ser or Thr of the β-subunit by means of the intramolecular cleavage. The second event results in further cleavage at the second scissile bond and finally releases the spacer peptide (5.Li Y. Chen J. Jiang W. Mao X. Zhao G. Wang E. Eur. J. Biochem. 1999; 262: 713-719Crossref PubMed Scopus (68) Google Scholar, 6.Lee Y.S. Kim H.W. Park S.S. J. Biol. Chem. 2000; 275: 39200-39206Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The study for the autoproteolytic mechanism of intermolecular cleavage was carried out in several precursor structures of N-terminal hydrolase. The structures of the GA and PS precursors were determined, and the autoproteolytic mechanisms for intramolecular cleavage by the Thr residue were proposed (4.Ditzel L. Huber R. Mann K. Heinemeyer W. Wolf D.H. Groll M. J. Mol. Biol. 1998; 279: 1187-1191Crossref PubMed Scopus (105) Google Scholar, 7.Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Interestingly, two Ntn hydrolases (GA and PS) proceed to autoproteolysis along different paths even if the same amino acid (Thr) was used as a key nucleophile for autoproteolysis. This means that a water molecule enhances the nucleophilicity of the Thr Oγ at the +1 position in PS, although the Asp-151 carboxylate at the -1 position promotes the nucleophilicity of the Thr-152 at the +1 position in GA. The intramolecular proteolysis proceeds to a N → O or N → S acyl shift (8.Tikkanen R. Riikonen A. Oinonen C. Rouvinen J. Peltonen L. EMBO J. 1996; 15: 2954-2960Crossref PubMed Scopus (84) Google Scholar, 9.Schmidtke G. Kraft R. Kostka S. Hemklein P. Frommel C. Lowe J. Huber R. Kloetzel P.M. Schmidt M. EMBO J. 1996; 15: 6887-6898Crossref PubMed Scopus (160) Google Scholar, 10.Guan C. Liu Y. Shao Y. Cui T. Liao W. Ewel A. Whitaker R. Paulus H. J. Biol. Chem. 1998; 273: 9695-9702Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and leads to an ester or thioester that is subsequently hydrolyzed to carboxyl and amino groups. The resulting N-terminal Ser, Thr, or Cys residue becomes exposed to the solvent to participate in the nucleophilic catalytic center for the Ntn hydrolases (2.Murzin A.G. Curr. Opin. Struct. Biol. 1996; 6: 386-394Crossref PubMed Scopus (224) Google Scholar, 7.Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11.Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (547) Google Scholar). The structures of CA from Pseudomonas diminuta KAC-1 (CAD) and a precursor CAD were determined (12.Kim Y. Yoon K.H. Khang Y. Turley S. Hol W.J.G. Structure (Lond.). 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Also, the mechanism of the intramolecular cleavage of the scissile peptide bond at the N-terminal side of the catalytic Ser-1β 2The notation of the amino acid sequence in precursor CAD is identical to the CAD up to residue 158α (12.Kim Y. Yoon K.H. Khang Y. Turley S. Hol W.J.G. Structure (Lond.). 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The α and β subunits are indicated by attaching α and β, respectively, to the end of the residue number. In addition, the spacer residue is represented by an “s” at the end of a residue number, such as 159s. The spacer sequence of the precursor CAD is from residue 159s to 169s in this structure (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14.Kim S. Kim Y. J. Biol. Chem. 2001; 276: 48376-48381Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). was proposed. Nucleophilic attack on the carbonyl carbon at the N-terminal side by the OG atom of Ser-1β results in ester formation between the carbonyl carbon of Gly-169s (amino acid immediately before the nucleophilic Ser-1β) and the OG atom of Ser-1β. Initially, the hydroxyl of Ser-1β is assisted by the conserved water, which is stabilized by four hydrogen bonds in a pseudo-tetrahedral geometry, and may accept a proton from the hydroxyl group. As a result nucleophilic attack takes place on the main chain carbonyl carbon of the Gly-169s. The detailed pattern of autoproteolysis in the precursor CA is some-what different from the previously determined precursor structures of the Ntn hydrolase families such as GA (7.Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and PS (4.Ditzel L. Huber R. Mann K. Heinemeyer W. Wolf D.H. Groll M. J. Mol. Biol. 1998; 279: 1187-1191Crossref PubMed Scopus (105) Google Scholar) in terms of both the type of the nucleophilic residue and the reaction pattern. A previous structural study of the precursor, CAD, revealed that conserved water (present in both the precursor CAD and the mature CAD) plays an important role in assisting the OG atom of Ser-1β so that the nucleophilic OG atom can carry out an attack on the scissile carbonyl carbon of Gly-169s (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The study proposed that the bound water assists the autocatalytic proteolysis of intramolecular cleavage. However, the role of bound water has not been fully examined due to insufficient experimental data. This study examined the role of bound water. A mutant CA that had lost the capability of autocatalytic proteolysis and remained a precursor enzyme was obtained. The structure of the mutant CA was determined at a 2.5-Å resolution. The precursor structure that was disabled in the autocatalytic proteolysis lost the bound water from the active site. A comparison of the active sites between the two different precursors with and without the autocatalytic proteolysis provided data that showed that the absence of bound water abolished the capability of carrying out the autoproteolytic intramolecular cleavage from the precursor CA. Also, the disability of the intramolecular proteolysis was the result of a mutation of the residues, causing a conformational distortion to the active site. This study provides the first evidence that a bound water molecule can play a critical role in initiating the beginning of intramolecular cleavage in the post-translational modification of the precursor enzyme. Crystallization—The mutant CAD of the F177β residue to Pro (F177βP), 3The mutation of Phe-177β to Pro is abbreviated to Phe177βP. The same notations are used for the other mutants accordingly. with no capability of autocatalytic proteolysis (14.Kim S. Kim Y. J. Biol. Chem. 2001; 276: 48376-48381Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar)) from P. diminuta KAC-1 (GenBank™ accession number AF251710; Ref. 16.Kim D.-W. Kang S.-M. Yoon K.-H. J. Microbiol. 1999; 37: 200-205Google Scholar), was prepared by site-directed mutagenesis and subcloned into Escherichia coli BL21(DE3) using the overexpression vector pET24d(+). The protein was then purified using His-tag affinity chromatography. The precursor CAD concentration was 10 mg ml-1 in a storage buffer (50 mm sodium phosphate, pH 7.0, and 150 mm NaCl). The F177βP precursor CAD crystals grew overnight at 21 °C from hanging drops containing 3 μl of a protein solution (10 mg ml-1 precursor CAD, 50 mm sodium phosphate, pH 7.0, and 150 mm NaCl) and 3 μl of a reservoir solution (16%(w/v) polyethylene glycol 8000, 10 mm dithiothreitol, 200 mm magnesium acetate, and 100 mm sodium cacodylate, pH 7.0) by the vapor diffusion method against a 500-μl reservoir solution. The crystals of the F177βP precursor CAD grew under quite similar conditions to the native CAD crystals (12.Kim Y. Yoon K.H. Khang Y. Turley S. Hol W.J.G. Structure (Lond.). 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). They belong to the same space group, P41212 (Table I), with almost identical unit cell dimensions (12.Kim Y. Yoon K.H. Khang Y. Turley S. Hol W.J.G. Structure (Lond.). 2000; 8: 1059-1068Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). There is a single chain precursor of 77 kDa per asymmetric unit and a 57% solvent content. The crystals were transferred to a cryo solution (containing 18% (w/v) of polyethylene glycol 8000, 10% (w/v) glycerol, 200 mm magnesium acetate, 10 mm dithiothreitol, 100 mm sodium cacodylate, pH 7.0) for 3 days before being flash-cooled in a 100 K gaseous nitrogen stream.Table ISummary of the crystallographic dataF177BP precursor CADDiffraction data statisticsSpace groupP41212Unit cell dimensions (Å)a = b = 73.7, c = 383.7Wavelength (Å)0.91005Resolution rangeaValues within the parentheses are for the last shell of data (Å)20—2.5 (2.59—2.50)Completeness (%)95.7 (99.9)Reflections, total242735Reflections, unique37824Rsym (%)bRsym = Σ | I — 〈1〉 | /ΣI10.2 (47.6)I/σ8.2 (5.8)X-ray sourcePAL 6MXRefinement statisticsResolution (Å)20-2.5RcrystcRcryst = Σ∥Fobs | — | Fcalc∥/Σ | Fobs |. All data were used with no sigma cutoff22.3RfreedRfree = Σ∥Fobs | — Fcalc∥/Σ | Fobs |, where Fobs are test set amplitudes (3.5%), not used in refinement. All data were used with no sigma cutoff26.7No. of non-H atoms Protein5374 Water225r.m.s.d.eRoot mean square deviations Bonds (Å)0.0072 Angles (°)1.35Average B-factors37.6a Values within the parentheses are for the last shell of datab Rsym = Σ | I — 〈1〉 | /ΣIc Rcryst = Σ∥Fobs | — | Fcalc∥/Σ | Fobs |. All data were used with no sigma cutoffd Rfree = Σ∥Fobs | — Fcalc∥/Σ | Fobs |, where Fobs are test set amplitudes (3.5%), not used in refinement. All data were used with no sigma cutoffe Root mean square deviations Open table in a new tab Data Collection—The data set for the F177βP precursor CAD crystal was collected to a resolution of 2.5 Å at the Pohang Accelerator Laboratory 6MX beamline from frozen crystals using a wavelength of 0.91005 Å. The data were indexed and integrated using DENZO and scaled by SCALEPACK (17.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38592) Google Scholar). Refinement—The structure of the F177βP precursor CAD yielded an excellent initial crystallographic model that is based on the structure of the S1βA precursor CAD (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Therefore, only minor adjustments were needed during the course of refinement. Fo - Fc difference Fourier maps provided an excellent guide for locating the residues Fig. 1). The modeled residues were refined using the program O (18.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) based on the structure of the S1βA precursor CAD. All of the crystallographic refinements were carried out using the CNS program (19.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar) with a maximum-likelihood refinement. The model geometry was confirmed by PRO-CHECK (20.Laskowski R.A. MacArthur M.W. Moss D.S. Thorton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The structure of the F177βP precursor CAD is quite similar to that of the S1βA precursor CAD (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) except for the mutation sites, the deviations in the Cαs of the main chain backbone, and the residue conformation of the active site. Table I shows the data and refinement statistics. The figures were generated by MOLSCRIPT (21.Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), BOBSCRIPT (22.Esnouf R.M. J. Mol. Graph. Model. 1997; 15 (112-113): 132-134Crossref PubMed Scopus (1794) Google Scholar), and RASTER3D (23.Merritt E. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3878) Google Scholar). Site-directed Mutagenesis of CAD Yields a Different Status of Intramolecular and Intermolecular Cleavage—The CAD precursor of the wild-type spontaneously conducts intramolecular cleavage by autocatalytic proteolysis at the scissile peptide bond between the Gly-169s and Ser-1β residues upon folding after protein synthesis. Subsequently, the processed form of CAD performs intermolecular cleavage at the peptide bond between the Gly-158α and Glu-159s residues (3.Lee Y.S. Park S.S. J. Bacteriol. 1998; 180: 4576-4582Crossref PubMed Google Scholar, 13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 14.Kim S. Kim Y. J. Biol. Chem. 2001; 276: 48376-48381Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Many mutants of the active site residues were examined to determine whether they are properly post-translated to an active heterodimeric form of CAD. Some of the active site mutants did not undergo proper intramolecular or intermolecular cleavage (Fig. 2, a and b). In particular the mutation of Ser-1β to Ala (S1βA) completely lost its intramolecular autoproteolytic activity (24.Kwon T.H. Rhee S. Lee Y.S. Park S.S. J. Struct. Biol. 2000; 131: 79-81Crossref PubMed Scopus (11) Google Scholar) because the OG atom from the side chain of Ser-1β plays an important role in nucleophilic attack at the scissile peptide bond. The 77-kDa size of the S1βA protein, which contains the α-subunit, spacer, and β-subunit, appears as a single band that corresponds to the non-processed CAD (Fig. 2a). The S1βA precursor CAD could represent a wild-type precursor CAD with a proper active site conformation that would carry out autocatalytic intramolecular cleavage (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) once the mutated Ala residue at the β1 position is modeled to Ser. This is because the overall conformation of the precursor S1βA CAD structure is similar to the mature CAD structure. In fact, a r.m.s. deviation of 0.18 Å is obtained after superimposing the 672 common Cα atoms to a wild-type mature CAD (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Furthermore, the Cαsof the nine active site residues of the mature CAD, which directly interacted with the substrate, glutaryl-7-aminocephalosporanic acid, are superimposed onto the corresponding Cα atoms of the S1βA precursor CAD with a r.m.s deviation of 0.22 Å (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). One of the most interesting mutations is F177β to Pro (F177βP). The intramolecular cleavage is interrupted even though it possesses a catalytic nucleophile for intramolecular cleavage, Ser-1β (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The F177βP mutant is believed to be a conformational mutant; therefore, it cannot undergo nucleophilic attack at the scissile peptide for intramolecular cleavage. Other mutants show various stages of intramolecular and intermolecular cleavage (Fig. 2, a and b). The Ser-1β to Cys-1β (S1βC) mutation undergoes intramolecular cleavage, but its intermolecular cleavage is interrupted (Fig. 2, a and b). The mutations of the Tyr-33β residues show three different stages of processing in intermolecular cleavage (Fig. 2a). The Y33βF processes are normal to the mature heterodimeric CAD with the right-sized α and β subunits. Y33βS completes the intramolecular cleavage, but the intermolecular cleavage is interrupted. Y33βI accomplishes the intramolecular cleavage in part so that it produces mixed bands of sα and α (Fig. 2a). Four of the five Ser-152α mutants can normally undergo intramolecular and intermolecular cleavage to produce mature CADs, but only the intermolecular cleavage of the S152αP mutant is interrupted (Fig. 2b). Only the L24βG mutant of the three L24β mutations proceeds to a mature CAD, whereas, L24βF can undergo partial intermolecular cleavage, and the L24βR mutant cannot undergo intermolecular cleavage (Fig. 2b). It appears that the S152αP, L24βF, and L24βR mutants contain a trace amount of the precursor forms of the 77-kDa size, as shown in the SDS-PAGE (Fig. 2b). They all contain bulky side chains, but further investigation may be required to determine the relationship with the intermolecular cleavage. The F177βP mutant remains a non-processed precursor (the F177βP precursor CAD); therefore, its structure is very interesting for understanding the intramolecular cleavage from a mechanistic point of view. The conformational or functional moieties in the F177βP mutant may be damaged due to its mutation so that it cannot accomplish the catalytic action of intramolecular cleavage. For that reason, the structure of the precursor F177βP CAD was determined at a 2.5-Å resolution, and the structure was compared with that of the structure of the S1βA precursor CAD. Structure Determination of F177βP Precursor CAD—The structure of the F177βP precursor CAD was determined at a 2.5-Å resolution (Table I). In the course of the structure refinement, the Fo - Fc difference Fourier map, with neighboring residues around P177β and Ser-1β that were omitted from the phase calculation (Fig. 1, see also Fig. 5, a and b), showed a clear positive density for these sequence regions before adding any information of backbone residues in the unbiased map. The residues were easily adjusted into the Fo - Fc difference maps using the O program (18.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). They were then refined using CNS (19.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar) to an Rcryst of 22.3% and an Rfree of 26.7% (Table I). The S1βA precursor CAD was determined previously at a 2.5-Å resolution. It was refined to good geometry (Rcryst of 20.3%, Rfree of 23.7%) (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The space group of the F177βP crystal was P41212, which is the same as the S1βA precursor CAD. Therefore, the structure determination was straightforward and guided by the S1βA precursor CAD structure. The overall conformation of the F177βP structure was refined using the CNS program (19.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16978) Google Scholar). The neighboring regions of P177βP and Ser-1β were manually corrected based on the Fo - Fc difference maps using the O program (18.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The overall conformation of the F177βP precursor CAD was quite similar to the S1βA precursor CAD. However, the Cαs of the F177βP precursor CAD was not superimposed onto those of the S1βA precursor CAD as much as the S1βA precursor CAD was superimposed onto those of the mature CAD (Fig. 3). The r.m.s. deviation for the 62 Cαs of the active site pockets between the F177βP precursor CAD and the S1βA precursor CAD was 0.573 Å, whereas the r.m.s. deviation of the 51 Cαs between the S1βA precursor CAD and the mature CAD was 0.125 Å (11 residues of the spacer peptide were not used for the superposition) (Fig. 3). The superposition among the three CADs (mature CAD, S1βA, and F177βP) showed that the S1βA precursor CAD was similar to the mature CAD. However, the F177βP precursor CAD was considerably deviated from both the mature CAD and the precursor S1βA structures. This comparison suggests that the active site of the F177βP precursor CAD was significantly distorted from that of the wild-type CAD. It is clear that the mutation of F177β to Pro imposes much strain on the main chain backbone of F177βP. Therefore, it induces significant conformational changes in the active site. This conformational distortion is leveraged into a non-processed precursor CAD for intramolecular cleavage, resulting in the F177βP precursor CAD. It will be interesting to investigate how one amino acid substitution is linked to abolishing the intramolecular cleavage in the post-translational modification. Conceivably, if these conformational mutations occur in nature, then they will have an influence upon the selection procedure in the post-translational modification. Model of Autocatalytic Proteolysis in Intramolecular Cleavage—The structures of the S1βA precursor CAD and the mature CAD led us to previously propose the model of the auto-catalytic intramolecular cleavage (Fig. 4, a and b) (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). A nucleophilic attack on the carbonyl carbon of Gly-169s by the OG atom of Ser-1β resulted in an ester formation between the carbonyl carbon of Gly-169s and the OG atom of Ser-1β. Previously, the hydroxyl of Ser-1β was assisted by the conserved water (WAT1 in Figs. 4, a and b, and 5a) and by the four hydrogen bonds in pseudo-tetrahedral geometry. WAT1 may enable the hydroxyl of Ser-1β to be precisely positioned and assist the polarization of the hydroxyl for nucleophilic attack. A tetrahedral intermediate can form after the OG atom of Ser-1β carries out the nucleophilic attack on the main chain carbonyl carbon of Gly-169s. Subsequently, the resulting oxyanion may move toward WAT2 (Figs. 4b and 5a) to be stabilized by the hydrogen bonds from the plausible oxyanion hole, which is not formed in the nucleophilic attack stage, but it may form in the transition state, consisting of WAT2 (Fig. 5a) and the main chain NH of His-23β. The oxyanion intermediate may then collapse, resulting in a shift in the linkage of the amide bond to an ester bond (N → O acyl shift) (3.Lee Y.S. Park S.S. J. Bacteriol. 1998; 180: 4576-4582Crossref PubMed Google Scholar, 25.Saarela J. Laine M. Tikkanen R. Oinonen C. Jalanko A. Rouvinen J. Peltonen L. J. Biol. Chem. 1998; 273: 25320-25328Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Finally, the conserved water, WAT1, may carry out a second nucleophilic attack on the carbonyl carbon of the newly formed ester intermediate, which would result in a free N-terminal Ser-1β and a carboxylate of Gly-169s (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In the proposed model the conserved water, WAT1, played a key role in assisting the critical nucleophilic residue of Ser-1β (13.Kim Y. Kim S. Earnest N.T. Hol G.J.H. J. Biol. Chem. 2002; 277: 2823-2829Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The hydroxyl of Ser-1β was indispensable in the autocatalytic intramol" @default.
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• W2078833893 date "2004-01-01" @default.
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• W2078833893 title "A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase" @default.
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