Functional Characterization and Structural Modeling of a Novel Glycine Oxidase from Variovorax paradoxus Iso1

ABSTRACT

The N-acyl-d-amino acid amidohydrolase (N-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of N-acyl-d-amino acids to yield consistent d-amino acids. A putative FAD-binding glycine/d-amino acid oxidase was located immediately upstream of the N-d-AAase gene. The gene encoding this protein was cloned into Escherichia coli BL21 (DE3)pLysS and overexpressed at 25°C for 6 h with 0.5 mM isopropyl β-d-1-thiogalactopyranoside induction. After purification, the tag-free recombinant protein was obtained. The enzyme could metabolize glycine, sarcosine, and d-alanine, but not l-amino acids or bulky d-amino acids. Protein modeling further supported these results, demonstrating that glycine, sarcosine, and d-alanine could fit into the pocket of the enzyme's activation site, while l-alanine and d-threonine were out of position. Therefore, this protein was proposed as a glycine oxidase, and we designated it VpGO. Interestingly, VpGO showed low sequence similarity to other well-characterized glycine oxidases. We found that VpGO and N-d-AAase were expressed on the same mRNA and could be transcriptionally induced by various N-acetyl-d-amino acids. Western blotting and zymography showed that both proteins had similar expression patterns in response to different types of inducers. Thus, we have identified a novel glycine oxidase that is co-regulated with N-d-AAase in an operon, and metabolizes N-acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1.

IMPORTANCE The Gram-negative bacterium Variovorax paradoxus has numerous metabolic capabilities, including the association with important catabolic processes and the promotion of plant growth. We had previously identified and characterized an N-acyl-d-amino-acid amidohydrolase (N-d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both in vitro and in vivo) another potential gene found in the promoter region of this N-d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with N-d-AAase. The structural basis for its FAD-binding potential and substrate stereo-specificity were also elucidated. This study first reported a novel glycine oxidase from V. paradoxus. We believe that our study makes a significant contribution to the literature, because this enzyme has great potential for use as an industrial catalysis, as a biosensor, and in agricultural biotechnology.

INTRODUCTION

d-amino acids play important roles in many physiological functions. Microorganisms can use exogenous d-amino acids as nutrition and energy sources. d-alanine and d-glutamate are components of the bacterial cell wall. In bacteria, d-amino acids are critical constituents of peptidoglycan, which can protect the cell walls from protease and even some antibiotics. In addition, some bacteria secrete d-amino acids to the environment and incorporate them into the cell walls for stress tolerance (1). Besides their important physiological roles, d-amino acids are also important in the pharmaceutical application for making antibiotics and anti-microbial peptides (2). In eukaryotes, d-amino acids play an important role in regulating many processes, including learning and memory formation, hormone secretion, arterial hypertension, and intracellular osmotic pressure (3–7). Abnormal regulation of d-amino acid levels can lead to pathological changes. For example, decreased levels of d-serine result in lower activity of N-methyl-d-aspartate (NMDA) receptors, leading to the development of schizophrenia (8, 9).

The content of d-amino acids is regulated by the d-amino-acid oxidase (DAAO) (EC 1.4.3.3). Changes in DAAO have been found to correlate with various pathological conditions. DAAO is an FAD-containing enzyme that catalyzes the oxidative deamination of the d-isomers of neutral amino acids to produce α-keto acids and ammonia. The d-isomers of basic amino acids and glycine are also catalyzed at lower efficiency. This enzyme has been identified in many organisms, from microorganisms to humans (5). Because DAAO plays important physiological roles in the regulation of d-amino acid levels, it could be used in the production of building blocks for several pharmaceuticals (10). Cephalosporins are used in more than half of all antibiotics and are synthesized through the 7-aminocephalosporanic acid (7-ACA) intermediate. 7-ACA has been produced from the chemical hydrolysis of cephalosporin C; however, the yield is low. This method has been largely replaced by a two-step biocatalytic transformation using DAAO and glutaryl-7-aminocephalo-sporanic-acid acylase (11).

Glycine oxidase (GO) (EC 1.4.3.19) is a flavoenzyme containing non-covalently bound FAD (12, 13), and partially shares substrate specificity with both DAAO and sarcosine oxidase (SOX) (EC 1.5.3.1). GO differs from DAAO because it has preferences for glycine, sarcosine, d-pipecolate, and other select d-amino acids (14) to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide (12). The thiO gene of Bacillus subtilis encodes an FAD-dependent GO. This enzyme forms a homotetramer and is required for the biosynthesis of the thiazole moiety of thiamin pyrophosphate (15). Because GO can catalyze the oxidation of the primary and secondary amines of a variety of molecules, it has great potential for use in industrial catalysis, biosensors, and agricultural biotechnology (16–18).

N-acyl-d-amino-acid amidohydrolase (N-d-AAase) (EC 3.5.1.81) hydrolyzes the acyl group from N-acyl-d-amino acids, which are precursors during d-amino acid synthesis. This type of enzyme has been found in numerous bacterial genera, including Alcaligenes, Streptomyces, and Pseudomonas (19). In our lab, the gene encoding N-d-AAase from Variovorax paradoxus Iso1 was previously cloned and expressed, and the recombinant enzyme was subsequently purified and characterized (20). In addition, site-directed mutagenesis to enhance the N-d-AAase activity and stability of this enzyme was performed (21). Because of the high stereospecificity of N-d-AAase for its substrate N-acyl-d-amino acids, N-d-AAase can distinguish between enantiomers in N-acyl-dl-amino acid mixtures, and thus has the potential to be a stereoselective biocatalyst.

V. paradoxus is a Gram-negative bacterium that was isolated in 1969 and originally named Alcaligenes paradoxus (22). It has diverse metabolic capabilities to degrade many anthropogenic contaminants and promote plant growth (via plant growth-promoting rhizobacteria [PGPR], for example). In 1991, it was renamed Variovorax paradoxus (23) and its whole genome was sequenced in 2011 (24). Previously, we isolated one strain of V. paradoxus (called Iso1), and its N-d-AAase was identified and characterized (20). During isolation of the N-d-AAase gene, the DNA fragment that included the 5′ flanking region was also cloned and sequenced. We have been interested in a unique gene located in this upstream region of the N-d-AAase gene, which encodes a potential FAD-binding oxidoreductase. In this study, this V. paradoxus protein was characterized both in vivo and in vitro, and was identified as a GO. Furthermore, the structural basis of the FAD-binding potential and substrate stereospecificity of this GO from V. paradoxus Iso1 was elucidated.

RESULTS

Sequence analysis of a putative open reading frame (ORF) upstream of the N-d-AAase gene.

The N-d-AAase of V. paradoxus Iso1, which is responsible for d-amino acid production, has been reported previously (20). We cloned the flanking region of the N-d-AAase gene, and the sequences encoding several ORFs are presented in Fig. 1. The whole genome sequences of 3 V. paradoxus strains were found in the NCBI database, including B4 (25), S110 (24), and EPS. A racemase is usually contained within the N-d-AAase operon of these strains, and V. paradoxus EPS has 2 copies of N-d-AAase (Fig. 1; EPS1, 2). While Iso1 lacks a racemase in this operon, a putative upstream ORF of 1,251 bp in close proximity to the N-d-AAase gene exists only in Iso1 (Fig. 1). This putative protein encoded 416 amino acid residues with a predicted molecular mass of 44,998 Da.

FIG 1.

Comparisons of the flanking regions of N-d-AAase in various Variovorax paradoxus strains. TF is an abbreviation of transcriptional factor.

This putative protein was subjected to a BLASTP search to identify its potential biological function in V. paradoxus Iso1. The results are summarized in Table 1. The primary sequence of the protein is similar to that of an FAD-binding oxidoreductase of Comamonas composti (59% identity and 71% similarity), Aureimonas populi (56% identity and 68% similarity), Cupriavidus pauculus (55% identity and 70% similarity), and Hydrogenophaga borbori (56% identity and 70% similarity); a d-amino acid dehydrogenase of Acidovorax wautersii (56% identity and 70% similarity) and Ralstonia pickettii (54% identity and 67% similarity); and an amino acid dehydrogenase of Pseudomonas fluorescens (53% identity and 67% similarity). The alignment of these sequences is shown in Fig. 2. A consensus motif of GXGXXG(X)₁₇D was found in these putative proteins near the N-terminus. It has been reported that the FAD molecule and FAD-binding domain have a conserved Rossmann fold beta-alpha-beta motif, which serves as a dinucleotide-binding motif (26). The predicted FAD-binding motif in these proteins is indicated with black line on the bottom of the sequence in Fig. 2.

TABLE 1.

Comparison of the amino acid sequence similarity of glycine oxidase from Variovorax paradoxus and other species

Strain (Accession no.)	Amino acid residues	Amino acid		Gene annotation
		Identity (%)	Similarity (%)
Comamonas composti (WP_027014269)	416	59%	71%	FAD-binding oxidoreductase
Aureimonas populi (WP_209738819)	416	56%	68%	FAD-binding oxidoreductase
Acidovorax wautersii (KAF1019419)	414	56%	70%	d-amino acid dehydrogenase
Cupriavidus pauculus (WP_124686222)	421	55%	70%	FAD-binding oxidoreductase
Hydrogenophaga borbori (WP_116957470.1)	412	56%	70%	FAD-binding oxidoreductase
Ralstonia pickettii DTP0602 (AGW90528.1)	413	54%	67%	d-amino acid dehydrogenase
Pseudomonas fluorescens ABAC62 ( KTC43732.1)	411	53%	67%	Amino acid dehydrogenase

FIG 2.

Multiple sequence alignments of putative ORFs located upstream of N-d-AAase in Variovorax paradoxus Iso1 with other related sequences listed in Table 1. Amino acid sequences aligned using ClustalW2. The FAD-binding motif, motif 1 and 2 are depicted at the bottom of the sequences with a black line. Red backgrounds show strictly conserved residues; red letters indicate residues that are well conserved within a group according to a Raisler matrix; the remaining residues are shown in black. Residues conserved between groups are boxed.

This putative protein from V. paradoxus Iso1 was further analyzed using a conserved domain search (27). A DadA domain with an accession of COG0665, described as glycine/d-amino acid oxidase (deaminating), was identified with an E-value of 1.13e⁻³⁸. Thus, the above data suggest that this protein may function as a glycine/d-amino acid oxidase (GO/DAAO) and involve amino acid transport and metabolism in V. paradoxus Iso1.

Subcloning and optimal expression of recombinant GO/DAAO in Escherichia coli.

To examine the activity of this putative GO/DAAO enzyme, we cloned the gene into the pET21a expression vector for expression in E. coli BL21 (DE3)pLysS. Under IPTG induction, cells harboring pET-21a-GO (carrying the go/daao gene) expressed the recombinant protein with an observed molecular mass of 45 kDa, as assessed by anti-GO Western blotting (Fig. 3A). To optimize go/daao expression, different incubation temperatures and induction periods under fixed IPTG concentrations were tested. GO/DAAO showed the highest expression at 25°C after 6 h of induction (Fig. 3B). Under these conditions, various IPTG concentrations were used to induce protein expression. The data indicated that 0.5 mM IPTG could achieve maximum expression levels (Fig. 3C). Because this enzyme may function in the metabolism of amino acids, the addition of dl-amino acids in the culture might facilitate its expression. Consistent with our expectation, when different percentages of dl-glutamate were added to the cultures, the enzyme specific activity was increased, up to 1.5 to 2-fold (Fig. 3D).

FIG 3.

Optimal expression of recombinant GO/DAAO. (A) SDS-PAGE and Western blot of crude lysates from pET21a/E. coli (lane 1) and pET21a-GO/E. coli (lane 2) after 6 h of incubation at 25°C with 0.5 mM IPTG. Molecular mass standards (lane M). The arrow indicates recombinant GO/DAAO protein. (B) Effect of temperature and time on go/daao gene expression. Transformants were incubated at different temperatures and times with 0.4 mM IPTG, and the enzyme specific activity were assayed. (C) Effect of IPTG. The cells were cultured in 2YT medium at 25°C with different amounts of IPTG for 6 h, and the specific activity of GO/DAAO were assayed. (D) Effect of DL-Glu. The cells were cultured in 2YT medium with 0.5 mM IPTG plus dl-Glu at 25°C for 6 h. Plotted values are the average of triplicate experiments ± standard deviation.

Thus, the conditions producing the best expression of this recombinant GO in pET21a was: Preculture at 25°C for 20 h and then inoculate 2YT medium with 5 to 6% of the preculture. After the absorbance at 600 nm had reached approximately 0.6, IPTG (0.5 mM) and dl-glutamate (2%) were added and incubated for another 6 h. In whole cell lysates, the specific activity of this oxidase could reach 880 mU/mg, while E. coli harboring pET21a alone could only produce 30 mU/mg.

Substrate specificity and phylogenesis identify GO/DAAO as a glycine oxidase.

To better characterize the predicted GO from V. paradoxus Iso1, its substrate specificity was examined. To avoid any potential perturbation of enzyme activity from the His-tag, the gene was cloned into pET41a to create a GST-fusion recombinant protein. After purification with glutathione-linked beads, the GST tag was removed by enterokinase cleavage.

A broad spectrum of substrates was tested in the activity assay. This GO had the highest enzymatic activity toward glycine and relatively low activity toward d-alanine and sarcosine. However, the enzyme had no apparent activity toward N-acetyl d/l-methionine and the other d-amino acids, and l-amino acids tested (Table 2). The major difference between DAAO and GO is that GO possesses activity toward sarcosine, whereas DAAO does not. Relative to its activity toward glycine, this GO had 32% activity toward sarcosine and 40% activity toward d-alanine. These data demonstrate that this enzyme is a GO, which is a deaminating enzyme that oxidizes primary amines and small d-amino acids. We have designated it as VpGO in subsequent sections. The GenBank accession number for VpGO is MZ199178.

TABLE 2.

The substrate specificity of VpGO

	VpGO
Substratea	Sp actb (mU/mg)	Relative activity (%)
Gly	2760 ± 10	100 ± 0.36
Sarcosine	890 ± 50	32.2 ± 5.6
d-Ala	1110 ± 20	40.2 ± 1.8
d-Thr	NDc	ND
d-Arg	60 ± 3	2.17 ± 5
d-Met	ND	ND
l-Ala	ND	ND
l-Met	ND	ND
N-acetyl-d-Met	ND	ND
N-acetyl-l-Met	ND	ND

^aSpecific activities of d-Glu, d-Asp, d-Lys, d-Tyr, d-Asn, d-Leu, d-Trp, d-Phe, and d-Val were tested and not detected.

^bData are the means of experiments performed in triplicate. Shown as mean ± standard deviation.

^cND, not detected.

The amino acid sequence of VpGO was compared with that of other bacterial GOs, whose functions have been characterized previously (Fig. 4). Several GOs have been identified in bacteria, including B. subtilis (13), Bacillus licheniformis (28), Bacillus cereus (17), Geobacillus kaustophilus (29), Pseudoalteromonas luteoviolacea (30), Pseudomonas putida (31), Bradyrhizobium jicamae, and Bradyrhizobium elkanii (18). The amino acid sequences of the GOs from the above-mentioned bacteria were retrieved. Phylogenetic relationships showed that VpGO was closest to the P. putida GO and farthest from the B. subtilis GO (Fig. 4). However, VpGO possessed a relatively low identity of 20 to 25% and similarity of 28 to 39% to these GOs.

FIG 4.

Phylogenetic tree of VpGO and other glycine oxidases. VpGO is compared to bacterial glycine oxidases with identified functions. The evolutionary distances are in the units of the number of amino acid differences per sequence. The entire positions with less than 70% site coverage were eliminated.

Molecular modeling of VpGO with ligands.

When we searched for structural homology using PSI-BLAST (32), VpGO showed 24% identity with GO from B. subtilis (Protein Data Bank (PDB) codes: 1RYI [33], 1NG3 [15], and 3IF9 [34]). Using B. subtilis GO (PDB: 3IF9) as a template, the molecular model of VpGO is shown in Fig. 5A. The interaction between FAD and VpGO was further analyzed using LIGPLOT (Fig. 5B). The interactions shown are those mediated by hydrogen bonds and by hydrophobic contacts. The green-colored dotted lines indicate hydrogen contacts between the cofactor FAD and 9 conserved amino acids of VpGO. Val14 and Asp33 positioned at the FAD-binding motif, others were positioned at motif 1 (Ser42, Ala46, and Leu48) and motif 2 (His378, Gly382, Leu383, and Thr384) (Fig. 2).

FIG 5.

Overall structure of VpGO and LIGPLOT analysis for the interaction of VpGO with FAD. (A) Ribbon diagram of the VpGO structure model. Helix are in red color and beta-strand are in blue. Cofactor FAD and substrate or inhibitor (glycine) are shown as a ball-and-stick model with green and cyan colors, respectively. (B) LIGPLOT analysis of FAD interaction is presented. Hydrogen bonds are indicated by green dotted lines between the atoms involved, and hydrophobic links are denoted by an red arc with spokes radiating toward the ligand atoms they link. Purple stick (FAD), orange stick (amino acid residues), red ball (O), black ball (C), and blue ball (N).

The active site of VpGO with FAD and glycine is also shown in the protein model (Fig. 6A and B). Different substrates were modeled based on modifications of hydroxyacetic acid. The smallest amino acid that does not have a chiral center (glycine) (Fig. 6C), a d-amino acid with a short side chain (d-alanine) (Fig. 6D), and N-methylglycine (also called sarcosine) (Fig. 6E) were positioned well in the catalytic center. In contrast, the l stereoisomer of alanine (Fig. 6F) and a d-amino acid with a short, branched side chain, d-threonine (Fig. 6G), had side chains that were mispositioned within the active site of the enzyme. Stereospecificity of VpGO model supports the idea that VpGO can only accommodate glycine and small d-amino acids.

FIG 6.

Structural comparison of different ligands in the binding site of VpGO. (A) The relative position of the enzyme, cofactor FAD and glycine are displayed on a translucent surface. (B) The section view is convenient to observe the existence of space of the FAD and glycine. FAD and glycine are shown as a stick model with cyan and green colors, respectively. Zoomed in view of ligands including (C) Glycine, (D) d-Alanine (yellow stick), (E) Sarcosine (gray stick), (F) l-Alanine (magenta stick) and (G) d-Threonine (orange stick) positioned in the activation cavity of VpGO.

Co-transcription of VpGO and N-d-AAase genes in V. paradoxus Iso1.

From the previous sequence prediction, the VpGO and N-d-AAase genes are potentially transcribed as operons (Fig. 7A). The potential promoter and terminator regions were first analyzed using bioinformatics described in Materials and Methods. Upstream of VpGO, a potential promoter sequence and ribosome binding site (RBS) were found (Fig. 7B). However, only an RBS was identified in the intergenic region (Fig. 7C). Similarly, a terminator was only found downstream of N-d-AAase but not in the intergenic region (Fig. 7D). This suggests that these 2 proteins may be encoded on the same transcript.

FIG 7.

Analysis of promoter and terminator of the VpGO and N-d-AAase operon. (A) Diagram of the potential operon. (B) and (C) Potential promoter region and ribosome binding site (RBS). (D) Potential terminator and its RNA structure. The colored image shows probable base-pairing in the stem-loop structure.

We used Northern blotting to verify this possibility. RNA was prepared from V. paradoxus Iso1 grown in LB or LB containing different inducers at 0.25%. Normalized RNA samples were separated by gel electrophoresis (Fig. 8A) and transferred to a nylon membrane. RNA transcripts were detected using probes for N-d-AAase and VpGO. Consistent with previous data (20), N-d-AAase mRNA could be induced with N-acetyl-d-Met but not with d-Met or N-acetyl-l-Met (Fig. 8B). Similarly, VpGO mRNA could only be transcribed in LB plus N-acetyl-d-Met (Fig. 8C). The data above show that the transcription of N-d-AAase and VpGO was induced specifically by N-acetyl-d-amino acids and not by N-acetyl-l-amino acids or d-amino acids. In addition, the size of the detected N-d-AAase and VpGO transcript was approximately 3 kb, which approximated the combined sizes of these 2 genes (Fig. 7A). Because N-acetyl-d-amino acids and not d-amino acids triggered the transcription of VpGO, this implies that VpGO and N-d-AAase are co-expressed to metabolize the same substrates.

FIG 8.

Northern blot analyses of the VpGO and N-d-AAase operon under different induction conditions. Variovorax paradoxus Iso1 cells were cultured in LB medium with different inducers at 0.25%. RNA was prepared and separated by (A) 1% formaldehyde agarose gel electrophoresis and transferred to a membrane for (B) Northern blotting with N-d-AAase or (C) VpGO probes. Lane 1, LB; lane 2, LB + d-Met; lane 3, LB + N-acetyl-d-Met; lane 4, LB + N-acetyl-l-Met; lane M, molecular mass marker (Knt).

Coordinate expression of VpGO and N-d-AAase proteins in V. paradoxus Iso1.

Since VpGO and N-d-AAase were induced concomitantly at the transcription level, we wished to examine whether their protein products were also expressed under similar conditions. Various N-acetyl-d-amino acids were added to the LB or minimal medium for this test (Fig. 9). Expression of N-d-AAase and VpGO was observed by Western blotting and zymography, respectively. We used zymography methods for visualizing VpGO based on substrate degradation. VpGO activity was analyzed in situ following electrophoresis, thus yielding the information on VpGO activity and the locations of active VpGO.

FIG 9.

Effects of different inducers on protein expression of N-d-AAase or VpGO. Variovorax paradoxus Iso1 cells were cultured in (A) LB or (B) minimal medium with different inducers at 0.25%. Cell lysates were separated by SDS-PAGE and N-d-AAase was detected by Western blot with anti-N-d-AAase antibody. VpGO activity and location was analyzed by zymography. Lane 1: control (no inducer); lane 2: N-acetyl-d-Ala; lane 3: N-acetyl-d-Met; lane 4: N-acetyl-d-Trp; lane 5: N-acetyl-d-Phe; lane 6: N-acetyl-l-Leu; lane 7: N-acetyl-dl-Leu; lane 8: N-acetyl-d-Leu. Molecular mass markers (kDa).

Consistent with the results of Northern blotting, N-d-AAase and VpGO showed similar expression patterns (Fig. 9). In LB medium, N-acetyl-d-Met, N-acetyl-d-Trp, N-acetyl-d-Phe, N-acetyl-dl-Leu, and N-acetyl-d-Leu induced both N-d-AAase and VpGO (Fig. 9A). In minimal medium, only N-acetyl-d-Phe, N-acetyl-dl-Leu, and N-acetyl-d-Leu induced both genes (Fig. 9B). The activity assays of N-d-AAase are also comparable with the Western blot. These results further support the hypothesis that the VpGO and N-d-AAase genes constitute an operon and are induced under similar conditions.

DISCUSSION

In this study, the glycine oxidase of V. paradoxus Iso1 was cloned, expressed, and identified. Sequence and structural comparisons revealed that the FAD-binding motif is conserved in this protein. The VpGO identified in this study is transcribed as an operon with N-d-AAase. The expression of this operon is induced by N-acetyl d-amino acids. While N-d-AAase can metabolize various N-acetyl d-amino acids (20), VpGO can metabolize only glycine, methylglycine (sarcosine), and d-Ala. VpGO has a higher preference for glycine than alanine but cannot metabolize l-amino acids, d-amino acids, or N-acetyl-d-amino acids. Protein homology modeling also supported the idea that neither l-amino acids nor d-amino acids with bulky side chains can fit into the activation cavity of VpGO.

GO has been demonstrated to be essential for the synthesis of the thiazole moiety of thiamine pyrophosphate in B. subtilis and other microorganisms (15). To our knowledge, this is the first study to demonstrate that N-d-AAase and VpGO are regulated in an operon in V. paradoxus. These 2 proteins may work together to metabolize N-acetyl-d-amino acids; N-d-AAase removes the N-acetyl group from d-amino acids and VpGO involves the deamination of various amines, as well as small d-amino acids to make the corresponding α-keto acids and ammonia/amine for other biosynthesis pathways. The results of this study contribute to the understanding of the metabolism of d-amino acids in bacterial physiology.

The reduced GO activity in E. coli likely occurs because GO acts on glycine and oxygen in the cell to produce excess hydrogen peroxide, which is hazardous. Another reason may be that d-alanine is an essential component of cell wall biosynthesis. When supplementing with dl-glutamate or dl-alanine to increase GO activity, dl-glutamate was more effective than dl-alanine in promoting VpGO overproduction. Induced cells grown in 2YT medium in the presence of 2% dl-glutamate had a relatively high GO specific activity of 880 mU/mg.

Sarcosine oxidase is a demethylating enzyme that cannot recognize glycine as a substrate. DAAO stereospecifically catalyzes the oxidative deamination of the amino group of d-amino acids to produce the corresponding α-keto acid and H₂O₂. DAAO poorly recognizes glycine and amino acid derivatives with a modified amino group as substrates, although it oxidizes d-alanine (13). Therefore, VpGO is neither a type of sarcosine oxidase nor a DAAO, and it is regarded as a deaminating glycine oxidase. Among the experimentally characterized GOs, VpGO is similar to P. putida GO, which shows a much narrower substrate specificity than GOs from B. subtilis and G. kaustophilus.

In terms of protein sequence, as far as we know, no protein from V. paradoxus strains that is similar to VpGO has been described to date. As shown in Fig. 1, the V. paradoxus strains reported hitherto have a putative FAD-dependent oxidoreductase, flavin reductase, and d-amino acid dehydrogenase that share only 28% to 37% identity with VpGO (24, 25, 35). Using PSI-BLAST with PDB for a structure search, VpGO was found to have similarity (24% identity) to the GO and sarcosine oxidase of B. subtilis. GOs are widespread in species of the genera Bacillus, Ochrobactrum, Pseudomonas, and Rhizobium. Although they typically possess the constant catalytic domain, their protein sequences are relatively variable (36). From these results, we report that VpGO is a novel GO that has low similarity to other known GOs.

Glyphosate is an organophosphate herbicide, and its application is a health risk concern. Gyphosate which is a derivative of glycine might be a target of GO at a low rate of activity. Recently, the GOs from Pseudomonas spp., Ochrobacterium sp., and Rhizobium sp. were reported to putatively possess low-level activity against the herbicide glyphosate (36). We might take advantage of using molecular tools to develop the enzyme usage of Gos for a variety of bioremediation, metabolism, and xenobiotic degradation issues.

MATERIALS AND METHODS

Enzymes, chemicals, plasmids, and bacterial strains.

d-amino acid oxidase from porcine kidney, horseradish peroxidase, catalase, and the enzyme substrate N-acetyl-d-methionine were purchased from Sigma-Aldrich, Calbiochem, and Merck, respectively. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. The Pfu DNA polymerase was purchased from Promega. The protein assay kit and 30% acrylamide/Bis-acrylamide solution were purchased from Bio-Rad. Plasmid pET21a and E. coli BL21a (DE3) were used as the protein expression vector and host, respectively. E. coli XL-1 blue was used for DNA manipulation and plasmid maintenance. The plasmid preparation and DNA gel purification were performed by using QIAprep spin miniprep kit and QIAquick gel extraction kit, respectively (Qiagen, GmBH). The bacterial strains and plasmids used in this study are listed in Table 3.

TABLE 3.

Bacterial strains and plasmids used in this study

Strain	Characteristics	Source
E. coli XL1-Blue	recA1 endA1 gyrA46 thi hsdR17 supE44 relA1 lac F’ (proAB lacIqZΔM15, Tn10[tetr])	Stratagene
E. coli BL21 (DE3)	F–ompT hsdSB (rB–, mB–) gal dcm (DE3)	Novagen
E. coli BL21 (DE3)pLysS	F–ompT hsdSB (rB–, mB–) gal dcm (DE3) pLysS(CamR)	Novagen
Variovorax paradoxus Iso1	N-acyl-d-amino acid amidohydrolase producing strain	20
Plasmids
pBK-damD4	Apr, a 12.3 kb plasmid, N -acyl-d-amino acid amidohydrolase gene (1,467-bp) and its flanking sequence from Variovorax paradoxus Iso1 cloned into pBluescript II KS (+) plasmid	20
pET21a	Apr, expression vector	Novagen
pET21-GO	Apr, 1,251-bp Vpgo gene amplified by PCR cloned into pET21a	This study
pET41a	Kmr, expression vector	Novagen
pET41a -GO	Kmr, 1,251-bp Vpgo gene amplified by PCR cloned into pET41a	This study

Bioinformatics.

To explore potential homologous proteins of the deduced gene product upstream of the N-d-AAase gene in V. paradoxus Iso1, the amino acid sequence was searched in BLASTP. Proteins with more than 53% identity were aligned using ClustalW2 (37). This novel protein from V. paradoxus Iso1 (named VpGO) was also analyzed using a conserved domain search (27).

When performing phylogenetic analysis of VpGO with other known GOs, the evolutionary history was deduced using the Neighbor-Joining method (38). The best tree is displayed. The percentage of duplicate trees in which the correlated taxa clustered together in the bootstrap test (1,500 duplicates) are provided next to the branches (39). The evolutionary distances were computed using the number of differences method (40). Evolutionary analyses were conducted using MEGA X (41).

The potential promoter region and ribosome binding site (RBS) were analyzed using the BDGP server (42). The potential terminator site was analyzed using the iTerm-PseKNC predictor (43), and the RNA structure was analyzed using the RNAfold server (44, 45).

Expression and purification of VpGO.

A 1.2 kb DNA fragment encoding the GO of V. paradoxus Iso1 was cloned into a pET21a or pET41a expression plasmid to produce recombinant VpGO proteins in E. coli (Table 3). A single colony of E. coli transformants was precultured at 37°C for 16 h in 3 mL Luria-Bertani (LB) broth (Difco) supplemented with 200 μg/mL ampicillin (Sigma). One to two percent of the overnight culture was inoculated into fresh medium and incubated at 150 rpm shaking. The incubation temperature, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, and IPTG induction time were optimized. VpGO with a glutathione S-transferase (GST) fusion tag was purified using the AKTA Prime Liquid Chromatography System (Pharmacia) with a GSTrap column (Amersham Pharmacia Biotech). After purification, the GST tag was cleaved by incubation with enterokinase (Novagen) at 22°C for 10 to 16 h.

Enzyme activity assay.

The method to measure the activity of N-d-AAase was described by Lin et al. (20). The reaction mixture contained the appropriate amount of enzyme and 25 mM N-acetyl-d-Met as the substrate. The reaction was allowed to proceed at 40°C and then stopped by heating to 100°C. The amount of d-methionine released was measured using the following colorimetric assay. Twenty microliters of DAAO (3 U/mL); 20 μL colorimetric solution comprising peroxidase (10 U/mL), 4-aminoantipyrine (0.04 μg/mL), and 0.8 μL phenol; and the appropriate volume of enzyme mixture were added to 50 mM Tris-HCl (pH 7.5) for a total volume of 200 μL. The reaction mixture was incubated at 25°C for 10 min and its absorbance at 520 nm was measured. Serial dilutions of 5 mM d-methionine were used for the standard curve. One unit was defined as the formation of 1 μmol d-methionine per min.

The activity of GO/DAAO was determined using spectrophotometry (46). An appropriate amount of enzyme was added to a reaction mixture containing 38 μg FAD, 100 mM d-alanine, and 300 μg bovine liver catalase in 57 mM potassium phosphate buffer (pH 8.0), and incubated at 37°C for 15 min. The 2,4-Dinitrophenylhydrazine was added ,and the incubation continued for another 5 min. Then, 150 μL trichloroacetic acid (TCA) was added to stop the reaction. The enzyme mixture was extracted with 0.7 mL ethyl acetate. The upper layer was transferred to a new tube and mixed well with 1.5 mL Na₂CO₃. The lower layer was mixed with the same volume of 1.5 N NaOH, and the absorbance at 520 nm was measured. Pyruvic acid (0.1–2 μmol) was used for the standard curve. One unit was defined as the formation of 1 μmol pyruvate per min.

For the VpGO substrate specificity assays, 0.2 mM d-amino acids, 50 μL buffer (50 mM Tris-HCl, pH 7.5), purified rVpGO, and colorimetric solution containing peroxidase (10 U/mL), 4-aminoantipyrine (0.04 μg/mL), and 0.8 μL phenol were used. The reaction mixture was incubated at 25°C for 10 min and the absorbance at 520 nm was detected. Serial dilutions of 0 to 0.1 μM H₂O₂ were used for the standard curve. The protein concentration was measured using the Bradford method, with bovine serum albumin (BSA) as a standard.

Homology modeling and docking.

Homology modeling was used to construct the model for VpGO. The amino acid sequence of VpGO was used to search for templates with PSI-BLAST (32) against the protein data bank (PDB). B. subtilis GO with FAD and hydroxyacetic acid (PDB code: 3IF9) (34) was selected as the structural template. Twenty best models were generated for GO using Modeller 9v8. Among the 20 models, one possessing the lowest discrete optimized protein energy (DOPE) score was selected and considered for further study. Based on the complex structure of B. subtilis GO with FAD and hydroxyacetic acid (PDB code: 3IF9) (34), FAD, and hydroxyacetic acid were docked into the binding sites of the VpGO model. The interaction between VpGO and FAD was further analyzed using LIGPLOT (47). The 3D structures of various substrates (glycine, d-alanine, sarcosine, l-alanine, and d-threonine) were modified from that of hydroxyacetic acid.

RNA preparation.

RNA was isolated using the modified hot-phenol method (48). Cultured cells were harvested and resuspended in 800 μL lysis buffer (33 mM sodium acetate, 0.5% SDS, 1 mM EDTA, pH5.5). Glass beads (0.8 g, 425 to 600 μm in diameter) were added to the cell suspension and vortexed for 2 min at 2,500 rpm. The samples were centrifuged, and the supernatants were transferred to a new tube and mixed with an equal volume of acid phenol (pH 5.5). After incubation at 68°C for 5 min, the samples were centrifuged. The aqueous layer was extracted again with an equal volume of chloroform and then mixed with 2 volumes of absolute ethanol and 1/10 volume of 3 M sodium acetate to precipitate the RNA. The RNA pellet was dried under vacuum and dissolved in diethyl pyrocarbonate (DEPC)-treated dH₂O.

Northern analysis.

The RNA samples were mixed with 3 volumes of NorthernMax Formaldehyde Load Dye (Ambion) and separated by electrophoresis in a 1% formaldehyde gel. The RNA was then transferred to a nylon membrane (Pharmacia) using the downward capillary method and fixed by UV cross-linking. The probe was synthesized using a PCR-DIG Labeling Kit (Roche). After hybridization at 60°C and washing, the membrane was incubated with CDP-Star chemiluminescent substrate (Tropix) for 15 min and then exposed to X-ray film.

Protein electrophoresis, Western blot, and zymography.

A full-length DNA fragment encoding the VpGO and N-d-AAase of V. paradoxus Iso1 was cloned into an expression plasmid to produce recombinant proteins in E. coli (Table 3). The proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane using a semidry transfer device (Pharmacia). N-d-AAase protein was detected with an anti-N-d-AAase antibody (1:20000; from Alcaligenes faecalis DA1). VpGO protein was detected by zymography. Zymography of VpGO was performed first by separation by 10% native PAGE on ice. Then, the gel was stained in 50 mL 0.35 M pyrophosphate buffer containing 0.356 g d-alanine, 50 μL FAD (19.1 mg/mL), and 250 μL iodonitrotetrazolium chloride (INT, 18.6 mg/mL) at 37°C.