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Deduced catalytic mechanism of D-amino acid amidase from Ochrobactrum anthropi SV3.
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PMID:  18421151     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
D-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes D-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of D-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Omega loop, and a ground state with a disordered Omega loop. Because the binding of the substrate facilitates ordering, this transition was regarded to be induced fit motion. The angles and distances of hydrogen bonds at Tyr149 Oeta, Ser60 Ogamma and Lys63 Nzeta revealed that Tyr149 Oeta donates an H atom to a water molecule in the substrate-bound state, and that Tyr149 Oeta donates an H atom to Ser60 Ogamma or Lys63 Nzeta in the ground state. Taking into consideration the locations of the H atoms of Tyr149 Oeta, Ser60 Ogamma and Lys63 Nzeta, a catalytic mechanism of DAA activity is presented, wherein a shift of an H atom at Tyr149 Oeta in the substrate-bound versus the ground state plays a significant role in the reaction. This mechanism explains well why acylation proceeds and deacylation does not proceed in the substrate-bound state.
Authors:
Seiji Okazaki; Atsuo Suzuki; Hidenobu Komeda; Yasuhisa Asano; Takashi Yamane
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-04-18
Journal Detail:
Title:  Journal of synchrotron radiation     Volume:  15     ISSN:  0909-0495     ISO Abbreviation:  J Synchrotron Radiat     Publication Date:  2008 May 
Date Detail:
Created Date:  2008-04-18     Completed Date:  2008-07-31     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  9888878     Medline TA:  J Synchrotron Radiat     Country:  Denmark    
Other Details:
Languages:  eng     Pagination:  250-3     Citation Subset:  IM    
Affiliation:
Department of Biotechnology, School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amidohydrolases / chemistry,  metabolism*
Catalysis
Hydrogen Bonding
Models, Molecular
Ochrobactrum anthropi / enzymology*
Chemical
Reg. No./Substance:
EC 3.5.-/Amidohydrolases; EC 3.5.1.-/D-amino acid amidase
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Journal ID (nlm-ta): J Synchrotron Radiat
Journal ID (publisher-id): J. Synchrotron Rad.
ISSN: 0909-0495
Publisher: International Union of Crystallography
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A full version of this article is available from Crystallography Journals Online.? International Union of Crystallography 2008
open-access: This is an open-access article distributed under the terms described at http://journals.iucr.org/services/termsofuse.html.
Received Day: 29 Month: 7 Year: 2007
Accepted Day: 29 Month: 11 Year: 2007
Print publication date: Day: 01 Month: 5 Year: 2008
Electronic publication date: Day: 18 Month: 4 Year: 2008
pmc-release publication date: Day: 18 Month: 4 Year: 2008
Volume: 15 Issue: Pt 3
First Page: 250 Last Page: 253
ID: 2394803
Publisher Id: ys5010
DOI: 10.1107/S0909049507064655
Coden: JSYRES
Publisher Item Identifier: S0909049507064655
PubMed Id: 18421151

Deduced catalytic mechanism of D-amino acid amidase from Ochrobactrum anthropi SV3 Alternate Title:Catalytic mechanism of D-amino acid amidase
Seiji Okazakia
Atsuo Suzukia
Hidenobu Komedab
Yasuhisa Asanob
Takashi Yamanea*
aDepartment of Biotechnology, School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan
bBiotechnology Research Center, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan
Correspondence: Correspondence e-mail: yamane@nubio.nagoya-u.ac.jp

1.  Introduction

D-Amino acids are important intermediates in the production of a number of chemicals, including pharmaceuticals, agrochemicals and food additives (Asano & L?bbeh?sen, 2000 ?).

Currently, D-amino acids and D-amino acids containing peptides are produced by enzymatic transformation (Schulze & Wubbolts, 1999 ?). Enzymatic transformation requires enzymes with high D-stereospecificity in order to approach the maximum theoretical yield (Schulze & Wubbolts, 1999 ?). D-Amino acid amidase (DAA) from the soil bacterium Ochrobactrum anthropi SV3 is an enzyme with this type of high D-stereospecificity, and catalyzes D-stereospecific hydrolysis of amino acid amides via bulky hydrophobic side chains to yield D-amino acid and ammonia (Asano et al., 1989 ?; Komeda & Asano, 2000 ?).

The crystal structures of DAA in the native form and in a complex with D-phenylalanine were determined and functionally characterized in our laboratory (Okazaki et al., 2007 ?). Several simulations suggested that Tyr149 O? is an adequate candidate for a general acid in the acylation step (Okazaki et al., 2008 ?). However, an overview of the catalytic mechanism of DAA functions, which include both acylation and deacylation, has remained unclear.

Previously we reported that substrate binding occurred in concert with a conformation change in the ? loop (residues 207?223; Okazaki et al., 2007 ?), and we regarded this motion as induced fit motion (Okazaki et al., 2008 ?). New concepts about pre-existing apo states, such as a conformational selection (Bosshard, 2001 ?) or selected fit mechanism (Wang et al., 2004 ?), have been proposed. However, the concept of induced fit, i.e. that the location of active site residues around a substrate stabilize a transition state and thereby promote the reaction (Koshland Jr, 1958 ?), remains very important.

In considering induced fit motion and geometries of hydrogen bonds at important residues in this work, we are able to speculate on the overall catalytic mechanism of DAA. The reaction mechanism that we propose provides a reasonable explanation of previously observed experimental results, including the fact that deacylation occurs at pH 6.8.


2.  Speculated H-atom shift of Tyr149 O? by induced fit motion

In the D-phenylalanine complex, subunits A?E belong to the substrate-bound state, and subunit F belongs to the ground state (Okazaki et al., 2007 ?). The distances of the shift between the substrate-bound state and the ground state in active site residues Ser60 O?, Lys63 N? and Tyr149 O? were 0.2, 0.6 and 1.2??, respectively (Fig. 1 ?). The r.m.s. deviation in atomic positions estimated via a Luzzati plot of the D-phenylalanine complex structures was 0.29??. This value suggests that the shifts of Lys63 N? and Tyr149 O? were significant. The extent of the shift of Tyr149 O? is the largest of the three; thus, it is plausible to consider that Tyr149 is important for induced fit motion. Tyr149 has been proposed to be a candidate for a general acid in acylation and a general base in deacylation (Okazaki et al., 2008 ?). The angles and distances of the hydrogen bonds related to Tyr149 O?, Ser60 O? and Lys63 N? are summarized for all subunits in the native and D-phenyl?alanine complexes (Table 1 ?). The data reveal that the Tyr149 C??Tyr149 O??Ser60 O? angle (greater than 130?) is far from the regular tetrahedron angle of 109.5?, which is suitable for donating an H atom to a hydrogen bond. The data also reveal that the Tyr149 C??Tyr149 O??H2O (less than 100.4?) and Tyr149 C??Tyr149 O??Lys63 N? angles (106?115?) are close to the regular tetrahedron angle. Taken together, these results suggested that the Tyr149 O? H atom in the substrate-bound state is donated to a hydrogen bond with a water molecule (namely, Z164; see Fig. 1 ?) or Lys63 N?. Additionally, the results of docking simulation using the MOE system (Version 2006.0801; Chemical Computing Group, Montreal, Canada) suggest that the leaving group NH2 of the substrate D-phenylalanine amide locates to the same position as the water molecule (Z164 in Fig. 1 ?) (Okazaki et al., 2008 ?). Together with the fact that acylation occurs in the substrate-bound state (Okazaki et al., 2007 ?), these findings lead us to propose that Tyr149 O? donates an H atom to the water molecule rather than to Lys63 N? in the substrate-bound state.

However, the results summarized in Table 1 ? suggested that Tyr149 O? forms hydrogen bonds to Ser60 O? or Lys63 N? in the ground state (subunit F in the D-phenylalanine complex). The Tyr149 C??Tyr149 O??Ser60 O? (104.2?) and Tyr149 C??Tyr149 O??Lys63 N? angles (99.1?) in subunit F in the D-phenylalanine complex seem to be suitable for hydrogen bonding. On the basis of these data alone, we cannot determine which residue interacts with the H atom of Tyr149 O? in the ground state. Both locations of the H atom of Tyr149 O? in the ground state are suitable for deacylation.

The hydrogen-bond geometry at Lys63 N? suggests that Lys63 N? may donate H atoms to Asn151 O?1 and Ala242 O (Table 1 ?). Lys63 N? is thought to be protonated under the conditions of crystallization (pH 6.8) (Okazaki et al., 2007 ?). Thus, on the basis of the geometries, one additional H atom from Lys63 N? may be donated to Tyr149 O?, rather than to Ser60 O?, in the substrate-bound state of the D-phenylalanine complex (Table 1 ?). For the ground state of the D-phenylalanine complex, however, there are no suitable candidate residues for acceptance of an H atom from Lys63 N? (Table 1 ?).

The angles and distances related to Ser60 O? suggest that Ser60 O? directs an H atom to Lys63 N? and not to Tyr149 O? (Table 1 ?). The effect is quite pronounced, particularly in the ground state.


3.  Proposed catalytic mechanisms

Reflecting the observations described above, we are able to propose a mechanism for catalysis of DAA at the crystallization pH (6.8) (Fig.?2 ?), wherein D-phenylalanine amide acts as a substrate.

In the unbound states, the proportion of molecules adopting the ground state is thought to be greater than the proportion adopting a substrate-bound state (Fig. 2 ?a) (Okazaki et al., 2007 ?). In this state, and considering the geometries of the residues (Table 1 ?), we propose that an H atom from Tyr149 O? is donated to Ser60 O? (Fig. 2 ?a). However, considering the angle and distances concerning Lys63 N? (Table 1 ?), it seems that an H atom of Lys63 N? is free and located at an intermediate position between Tyr149 O? and Ser60 O? (Fig. 2 ?a).

There are at least two possible ways for the initial transition from ground state to occur in terms of the catalytic mechanism of DAA (Fig. 2 ?a) versus the substrate-bound state (Fig. 2 ?b). One is via the traditional induced fit mechanism; in this case, the substrate first binds at the ground state and the complex then switches to the substrate-bound state. The other is via the conformational selection mechanism (Eisenmesser et al., 2005 ?) or selected fit mechanism (Boehr et al., 2006 ?), in which the substrate binds to the minor substrate-bound state conformation already present in solution.

The presence of an incoming substrate in the binding pocket should shift the ratio of structures in favor of the substrate-bound state by ordering the ? loop (Okazaki et al., 2007 ?) and bring about the relocation of Tyr149 O? in the substrate-bound state as described above (Fig. 2 ?b). We consider that adoption of the substrate-bound state lowers the activation energy necessary for the proceeding acylation step by creating an environment in which the transition state of acylation is stabilized (Okazaki et al., 2008 ?). After a substrate is incorporated into the catalytic cleft in the substrate-bound state, the general base Lys63 N? may deprotonate Ser60 O?, and the nucleophile Ser60 O? may then attack the amide group of the substrate, generating tetrahedral intermediates [light-green arrows in Fig. 2 ?(b)]. Subsequently, the leaving group of the tetrahedral intermediate should deprotonate the general acid Tyr149 O?, leading to release of NH3. In turn, Tyr149 O? may deprotonate Lys63 N?, and finally the acylation is completed [cyan arrows in Fig. 2 ?(b)]. Therefore, Lys63 and Tyr149 appear to be the most plausible candidates for the general base and acid, respectively, that act in acylation (Okazaki et al., 2008 ?).

In the substrate-bound state of the D-phenylalanine complex, acylation occurred but deacylation did not (Okazaki et al., 2007 ?). This may result from the specific position of the H atom of Tyr149 O? in the substrate-bound state. The OH group of Tyr149 in the substrate-bound state could easily donate an H atom to either the leaving group of the substrate or a water molecule: that is, the location of Tyr149 OH is well situated to participate in acylation (Fig.?2 ?b). In the next step, the positioning of Tyr149 OH in the substrate-bound state may prevent deprotonation of a nucleophilic water molecule and thus would not be well suited for deacylation [Fig. 2(d) ?; note that the NH3 group in Fig. 2 ?(d) is replaced by H2O in the D-phenylalanine complex]. After the release of NH3, Tyr149 OH should become positioned in a way that is well suited for deacylation: that is to say, the H atom of Tyr149 O? may be directed to either Ser60 O? or Lys63 N? (Fig. 2 ?e or 2 ?f).

The dynamics of transition from substrate-bound to ground state in DAA are still unclear; however, it is possible to speculate that the release of NH3 through the proposed NH3 channel (Okazaki et al., 2008 ?) can trigger a conformational transition from the substrate-bound to the ground state, and cause penetration of a nucleophilic water molecule in the catalytic center [Fig. 2 ?; from (d) to (e) or (f)]. Interestingly, the transition from the substrate-bound to the ground state generates movement of the phenyl group of Phe282, producing a space that permits a water molecule to access the catalytic center from the exterior. In other penicillin-recognizing proteins, such as DD-peptidase, class C ?-lactamase and EstB esterase, water molecules from the exterior are considered to be essential for deacylation (Wagner et al., 2002 ?). In fact, the catalytic clefts of DD-peptidase and extended spectrum class C ?-lactamase are maintained in the open form after substrate binding, which permits a water molecule to access the acyl enzyme (Negoro et al., 2007 ?).

We previously proposed that deacylation in subunit F of the D-phenylalanine complex is facilitated by extraction of an H atom from Tyr149 O? via a deprotonated His307 N?2 (Okazaki et al., 2007 ?). The Tyr149 C??Tyr149 O??His307 N?2 angle (122.2?) and the distance between Tyr149 O? and His307 N?2 (2.9??) in subunit F in the D-phenylalanine complex suggests that Tyr149 O? can donate an H atom to His307 N?2 as well as Ser60 O? and Tyr149 Lys63 N?. Therefore we could not exclude the possibility that His307 N?2 extracts the H atom of Tyr149 O?, as in the case of either Fig. 2 ?(e) or 2 ?(f).

To bring about a transition from the acyl-enzyme state to a free (deacylated) state, initially the general base Tyr149 O? must deprotonate a water molecule and the activated nucleophilic OH group must attack the carbonyl carbon of D-phenylalanine, generating a tetrahedral intermediate [light-green arrow in Fig. 2 ?(e) or 2 ?(f)]. Successively, a tetrahedral intermediate should deprotonate the general acid Tyr149 O?, leading to the release of D-phenylalanine [cyan arrow in Fig.?2 ?(e)]. Tyr149 O? is the stronger candidate for the general acid in deacylation, on the basis of the geometries observed for Tyr149 O? in the ground state (Table 1 ?). In contrast, Lys63 N? may be able to donate an H atom to Ser60 O? as the general acid in deacylation and subsequently Lys63 N? extracts an H atom from Tyr149 O? [cyan arrow in Fig. 2 ?(f)]. This model is supported from the viewpoint that the location of the H atom of Ser60 O? is predominantly at Lys63 N? in the ground state, as shown in Table 1 ?. Thus, the location of the H atom of Tyr149 O? in the ground state is considered to be an essential step prior to deacylation (Fig. 2 ?e or 2 ?f).


This work was supported in part by a research grant from the National Project on Protein Structural and Functional Analysis from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The synchrotron radiation experiments were performed at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal Nos. 2005B1793, 2005B0372, 2005A0841, 2004B0826 and 2004A0488).


References
Asano, Y. & L?bbeh?sen, T. L. (2000). J. Biosci. Bioeng.89, 295?306.
Asano, Y., Mori, T., Hanamoto, S., Kato, Y. & Nakazawa, A. (1989). Biochem. Biophys. Res. Commun.162, 470?474.
Boehr, D. D., McElheny, D., Dyson, H. J. & Wright, P. E. (2006). Science, 313, 1638?1642.
Bosshard, H. R. (2001). News Physiol. Sci.16, 171?173.
Eisenmesser, E. Z., Milet, O., Labeikovsky, W., Korzhnev, D. M., Wolf-Watz, M., Bosco, D. A., Skalicky, J. J., Kay, L. E. & Kern, D. (2005). Nature (London), 438, 117?121.
Komeda, H. & Asano, Y. (2000). Eur. J. Biochem.267, 2028?2035.
Koshland, D. E. Jr (1958). Proc. Natl Acad. Sci. USA, 44, 98?104.
Negoro, S., Ohki, T., Shibata, N., Sasa, K., Hayashi, H., Nakano, H., Yasuhira, K., Kato, D., Takeo, M. & Higuchi, Y. (2007). J. Mol. Biol.370, 142?156.
Okazaki, S., Suzuki, A., Komeda, H., Yamaguchi, S., Asano, Y. & Yamane, T. (2007). J. Mol. Biol.368, 79?91.
Okazaki, S., Suzuki, A., Mizushima, T., Komeda, H., Asano, Y. & Yamane, T. (2008). In preparation.
Schulze, Y. B. & Wubbolts, M. G. (1999). Curr. Opin. Biotech.10, 609?615.
Wagner, U. G., Petersen, E. I., Schwab, H. & Kratky, C. (2002). Protein Sci.11, 467?478.
Wang, C., Karpowich, N., Hunt, J. F., Rance, M. & Palmer, A. G. (2004). J. Mol. Biol.342, 525?537.

Article Categories:
  • Diffraction Structural Biology

Keywords: catalytic mechanisms, stereospecific hydrolysis, D-amino acid amidase.
Conference: 2nd International Symposium on Diffraction Structural Biology (ISDSB2007). . .

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