Figures and data in Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

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Tables

10 figures and 11 tables

Figures

Figure 1

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The reactions catalyzed by proline racemase (ProR), 4R-hydroxyproline 2-epimerase (4HypE), and *trans*-3-hydroxy-L-proline dehydratase (t3HypD) and the metabolic pathways in which they participate.

cHyp oxidase, Pyr4H2C deaminase, a-KGSA dehydrogenase, and ?¹-Pyr2C reductase belong to the D-amino acid oxidase (DAAO), dihydrodipicolinate synthase (DHDPS), aldehyde dehydrogenase, and ornithine cyclodeaminase (OCD) (or malate/L-lactate dehydrogenase 2 [MLD2]) superfamilies, respectively. Abbreviations: L-Pro: L-proline; D-Pro: D-proline; 5-AV: 5-aminovalerate; t4Hyp: *trans*-4-hydroxy-L-proline; c4Hyp: *cis-*4-hydroxy-D-proline; Pyr4H2C: ?¹-pyrroline 4-hydroxy 2-carboxylate; a-KGSA: a-ketoglutarate semialdehyde; a-KG: a-ketoglutarate; t3Hyp: *trans*-3-hyroxy-L-proline; ?²-Pyr2C: ?²-pyrroline 2-carboxylate; ?¹-Pyr2C: ?¹-pyrroline 2-carboxylate.

https://doi.org/10.7554/eLife.03275.003

Figure 2

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Sequence similarity networks (SSNs) for the PRS.

(A) The SSN displayed with an e-value threshold of 10^-55 (~35% sequence identity). (B) The SSN displayed with an e-value threshold of 10^-110 (~60% sequence identity).

https://doi.org/10.7554/eLife.03275.004

Figure 3

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The genome neighborhood network (GGN) for the PRS.

(A) The GNN displayed with an e-value threshold of 10^-20. The nodes are colored by the color of query nodes in the SSN (Figure 2A). The clusters are labeled with the UniProtKB/TrEMBL annotations. (**B–I**) Selected superfamily clusters from the GNN showing node colors. (B) D-proline reductase PrdA. (C) D-proline reductase, PrdB. (D) D-amino acid oxidase (DAAO). (E) Dihydrodipicolinate synthase (DHDPS). (F) Aldehyde dehydrogenase. (G) Ornithine cyclodeaminase (OCD). (H) Malate/L-lactate dehydrogenase 2 (MLD2). (I) Proline racemase.

https://doi.org/10.7554/eLife.03275.005

Figure 4

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Library of proline and proline betaine derivatives tested for ESI-MS screening.

These substrates were divided into four groups to avoid mass duplication.

https://doi.org/10.7554/eLife.03275.006

Figure 5

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Structures of members of the PRS.

(A) Structure of Q4KGU2 (locus tag: PFL_1412; cluster 2) with PYC illustrating the utilization of the carboxyl group to bridge the N-terminal amide backbone groups of two opposing a-helices. While In B9K4G4 (D) and B9JQV3 (C) the relative positions of residues that coordinate the prolyl nitrogen (Asp 232, His 90) are conserved His 90 is replaced by a Ser. (B) Structure of Q4KGU2 with t4Hyp illustrating the interactions Q4KGU2 with the 4-hydroxyl group and the relative positions of the two catalytic cysteine residues. (C) Structure of B9JQV3 (locus tag: Avi_0518, cluster 9) with t4Hyp illustrating the interactions of B9JQV3 with the 4-hydroxyl group of t4Hyp and the relative positions of the catalytic Ser (Ser 93, trans?cis) and Cys (Cys 236, cis?trans). (D) Structure of B9K4G4 (Avi_7022, cluster 3) with PYC illustrating the position of the catalytic Ser (Ser 90, dehydration), and the non-catalytic orientation of Thr 256 which replaces the Cys observed in Cys/Cys containing PRS members. In addition, the catalytic Ser (Ser 90) is positioned by hydrogen bonding interactions between the side chain of Asn 93 (shown) and the backbone nitrogen of Asn 93 (not shown). Based on this work, all ProR family members with a catalytic Ser at this position (including B9JQV3, determined here) are proposed to have this motif.

https://doi.org/10.7554/eLife.03275.012

Figure 6

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Sequence divergent members of the ornithine cyclodeaminase superfamily (OCDS) have been the assigned novel pyrroline-2-carboxylate reductase (Pyr2C reductase) function in this work.

(A) The OCDS SSN displayed at the e-value cutoff 10^-45 (~35% sequence identity). The Pyr2C reductase function is located in four clusters; these proteins are shown in large colored circles, labeled from 1 to 16, and color-coded by the colors of the PRS query sequences shown in Figure 2B. Proteins representing several previously characterized functions in the OCDS are shown by large diamonds, with borders in hotpink (L-alanine dehydrogenase [Schröder et al., 2004]), brown (ornithine cyclodeaminase [Goodman et al., 2004]), magenta (lysine cyclodeaminase [Gatto et al., 2006]), red (ketamine reductase [Hallen et al., 2011]), green (L-arginine dehydrogenase [Li and Lu, 2009]) and palegreen (tauropine dehydrogenase [Kan-No et al., 2005; Plese et al., 2008]), respectively. Their annotations are shown in italics. The diamonds with blue and olive borders are Pyr2C reductases recently characterized by Watanabe et al. (2014). (B) Kinetics data for the Pyr2C reductase activity for the 16 members of the OCDS shown in panel A using NADPH as the cosubstrate.

https://doi.org/10.7554/eLife.03275.014

Figure 7

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Mapping members of GNN clusters back to the SSN for the PRS.

(A) SSN for the PRS with cluster numbers. (B) D-amino acid oxidase (DAAO). (C) Dihydrodipicolinate synthase (DHDPS). (D) Aldehyde dehydrogenase. (E) Ornithine cyclodeaminase (OCD). (F) Malate/L-lactate dehydrogenase 2 (MLD2). (G) The color scheme for **B–F**.

https://doi.org/10.7554/eLife.03275.016

Figure 8

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Experimentally characterized enzymes reported by Swiss-Prot (small colored circles) and newly characterized in this work (large colored circles).

Colors match the color scheme in Figure 2B.

https://doi.org/10.7554/eLife.03275.017

Figure 9

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Demonstration of the 4HypE, 3HypE, and t3HypD reactions by ¹H NMR.

(A) ¹H NMR spectra of the 4Hyp substrate mixture in 25 mM Na⁺-phosphate buffer, pD 8, in D₂O (top) and 4Hyp mixture with **A3QFI1** (cluster 1, blue) showing 4Hyp epimerization (bottom). The red arrow indicates the proton at C2 for epimerization. The enzyme was stored in glycerol, so the spectra show resonances for glycerol between 3.4 and 3.7 ppm. (B) ¹H NMR spectra of the t3Hyp substrate mixture in 25 mM Na⁺-phosphate buffer, pD 8, in D₂O (top), t3Hyp mixture with D0B556 (cluster 3, light sky blue) showing 3Hyp epimerization (middle), and t3Hyp mixture with B9K4G4 (cluster 3, light sky blue) showing t3Hyp dehydration (bottom). The red arrow indicates the proton at C2 for epimerization; the green arrow indicates the proton at C3 for dehydration.

https://doi.org/10.7554/eLife.03275.018

Figure 10

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Representative ¹H NMR spectra for ?¹-pyrroline-2-carboxylate (?¹-Pyr2C) reductase activity.

(A) ¹H NMR spectrum of ?¹-Pyr2C substrate in sodium phosphate, pD 8.0, in D₂O. (B) ¹H NMR spectrum of Q7CVK1 (locus tag: Atu4676) incubated with ?¹-Pyr2C, NADPH, and the cofactor regeneration system of alcohol dehydrogenase (NADP⁺-dependent) and isopropanol in sodium phosphate, pD 8.0 in D₂O. (C) ¹H NMR spectrum of L-proline in 25 mM sodium phosphate, pD 8.0, in D₂O.

https://doi.org/10.7554/eLife.03275.019

Tables

Table 1

Mass spectroscopy screening results in D₂O. Hits were observed by mass shift for racemization/epimerization (+1) and dehydration (-17) for reactions performed

https://doi.org/10.7554/eLife.03275.007

Locus tag	UniProt	L-Pro	D-Pro	t4Hyp	c4Hyp	t3Hyp	cis-3-OH-L-Pro
Cluster 1: blue
Pden_4859	A3QFI1	0	0	+1	+1	0	0
Shew_2363	A9AQW9	0	0	+1	+1	0	0
Bmul_5265	A6WXX7	0	0	+1	+1	+1	+1
Oant_1111	D2QN44	0	0	+1	+1	+1	+1
Slin_1478	B9JHU6	0	0	+1	+1	+1	+1
Arad_8151	Q8FYS0	0	0	+1	+1	0	0
BR1792	A1BBM5	0	0	+ 1	+1	+1	+1
Cluster 2: red
A1S_1325	A3M4A9	0	0	+1	+1	+1	+1
Bamb_3550	Q0B9R9	0	0	+1	+1	+1	+1
BceJ2315_47180	B4EHE6	0	0	+1	+1	+1	+1
BMULJ_04062	B3D6W2	0	0	+1	+1	+1	+1
BTH_II2067	Q2T3J4	0	0	+1	+1	-17	0
CV_2826	Q7NU77	0	0	+1	+1	+1	+1
Csal_2705	Q1QU06	0	0	+1	+1	0	0
PFL_1412	A5VZY6	0	0	+1	+1	+1	+1
Pput_1285	Q1QBF3	+1	+1	+1	+1	+1	+1
Pcryo_1219	A3M4A9	0	0	+1	+1	+1	+1
XCC2415	Q8P833	0	0	+1	+1	+1	+1
Bmul_4447	A9AL52	0	0	+1	+1	+1	+1
ABAYE2385	B0VB44	0	0	+1	+1	+1	+1
BURPS1106B_1521	C5ZMD2	+1	+1	+1	+1	+1	+1
BURPS1710b_A1887	Q3JHA9	0	0	+1	+1	+1	+1
PA1268	Q9I476	0	0	+1	+1	0	0
Cluster 3: ligthskyblue
Pden_1184	A1B195	0	0	0	0	-17	0
SIAM614_28502	A0NXQ9	0	0	0	0	-17	0
Atu4684	A9CH01	0	0	+1	+1	-17	0
Avi_7022	B9K4G4	0	0	0	0	-17	0
Oant_0439	A6WW16	0	0	+1	+1	0	0
SM_b20270	Q92WR9	0	0	+1	+1	-17	0
BMEI1586	Q8YFD6	0	0	+1	+1	+1	+1
BR0337	Q8G2I3	0	0	0	0	-17	0
Cluster 5: navy
BC_0905	Q81HB1	0	0	+1	+1	-17	0
BCE_0994	Q73CS0	0	0	+1	+1	-17	0
BT9727_0799	Q6HMS9	0	0	+1	+1	-17	0
Cluster 9: orange
Avi_0518	B9JQV3	0	0	+1	+1	+1	+1
Atu0398	A9CKB4	0	0	+1	+1	+1	+1
RHE_CH00452	Q2KD13	0	0	+1	+1	+1	+1
Arad_0731	B9J8G8	0	0	+1	+1	+1	+1
Cluster 11: palegreen
Sros_6004	D2AV87	0	0	+1	+1	0	0
Cluster 12: olive
Bamb_3769	Q0B950	0	0	0	0	-17	0
Bmul_4260	A9AKG8	0	0	+1	+1	+1	+1
Cluster 16: salmon
Csal_2339	Q1QV19	0	0	+1	+1	0	0
Maqu_2141	A1U2K1	0	0	0	0	0	0
Cluster 17: lime
Rsph17029_3164	A3PPJ8	0	0	+1	+1	0	0
RSP_3519	Q3IWG2	0	0	+1	+1	0	0
Cluster 18: cyan
SIAM614_28492	A0NXQ7	0	0	+1	+1	0	0
SADFL11_2813	B9R4E3	0	0	+1	+1	0	0
SPOA0266	Q5LKW3	0	0	+1	+1	+1	+1
Cluster 22: steelblue
Spea_1705	A8H392	0	0	0	0	-17	0
Swoo_2821	B1KJ76	0	0	+1	+1	-17	0
Cluster 61:
Plim_2713	D5SQS4	0	0	+1	+1	+1	+1

Table 2

Kinetic constants for 3/4HypE and t3HypD activities of the screened PRS targets

https://doi.org/10.7554/eLife.03275.008

Cluster	Locus tag	UniProt	Function	k_cat [s^-1]	K_m [mM]	k_cat/K_M[M^-1s^-1]
1	Pden_4859	A1BBM5	4HypE	16 ± 2	25 ± 5	630
	Shew_2363	A3QFI1	4HypE	50 ± 6	12 ± 3	4000
	Bmul_5265	A9AQW9	3HypE	0.34 ± 0.03	- ^a	- ^a
	Bmul_5265	A9AQW9	4HypE	5.6 ± 0.5	11 ± 2	530
	Oant_1111	A6WXX7	3HypE	2.4 ± 0.2	31 ± 7	77
	Oant_1111	A6WXX7	4HypE	89 ± 2	7.1 ± 0.6	13000
2	BTH_II2067	Q2T3J4	t3HypD	17 ± 3	26 ± 9	660
	BTH_II2067	Q2T3J4	4HypE	40 ± 4	1.4 ± 0.4	28000
	CV_2826	Q7NU77	3HypE	30 ± 0.6	57 ± 4	520
	CV_2826	Q7NU77	4HypE	70 ± 7	6.8 ± 3	10000
	Pput_1285	A5VZY6	3HypE	4.8 ± 0.6	19 ± 5	250
			4HypE	26 ± 0.7	0.54 ± 0.08	48000
			ProR	2.8 ± 0.1	200 ± 20	14
	XCC2415	Q8P833	4HypE	28 ± 0.4	0.67 ± 0.05	42000
	XCC2415	Q8P833	3HypE	1.3 ± 0.07	15 ± 3	86
3	Pden_1184	A1B195	t3HypD	nd ^b	nd ^b	nd ^b
	SIAM614_28502	A0NXQ9	t3HypD	15 ± 0.9	7.8 ± 1	1900
	Atu4684	A9CH01	t3HypD	27 ± 1	4.2 ± 0.8	6300
	Atu4684	A9CH01	4HypE	0.40 ± 0.02	2.0 ± 0.3	200
	Avi_7022	B9K4G4	t3HypD	4.3 ± 0.4	15 ± 3	280
	Oant_0439	A6WW16	4HypE	0.064 ± 0.002	1.3 ± 0.2	49
	SM_b20270	Q92WR9	t3HypD	7.9 ± 0.2	3.8 ± 0.4	2100
	SM_b20270	Q92WR9	4HypE	0.089 ± 0.01	6.3 ± 2	14
	BMEI1586	D0B556	3HypE	0.085 ± 0.003	2.6 ± 0.4	33
	BMEI1586	D0B556	4HypE	0.082 ± 0.005	4.5 ± 1	18
	BR0337	Q8G2I3	t3HypD	17 ± 2	5.1 ± 2	3300
5	BCE_0994	Q73CS0	t3HypD	nd ^b	nd ^b	nd ^b
	BCE_0994	Q73CS0	4HypE	1.2 ± 0.03	3.2 ± 0.3	370
	BT9727_0799	Q6HMS9	t3HypD	23 ± 5	7.5 ± 3	3100
	BT9727_0799	Q6HMS9	4HypE	0.16	- ^a	- ^a
9	Avi_0518	B9JQV3	3HypE	0.75 ± 0.04	4.8 ± 0.9	160
	Avi_0518	B9JQV3	4HypE	1.3 ± 0.07	5.6 ± 0.5	230
	Atu0398	A9CKB4	3HypE	4.0 ± 0.6	25 ± 7	160
	Atu0398	A9CKB4	4HypE	0.86 ± 0.1	4.6 ± 2	190
	RHE_CH00452	Q2KD13	3HypE	0.94 ± 0.06	2.1 ± 0.7	450
			4HypE	1.9 ± 0.08	2.1 ± 0.3	880
11	Sros_6004	D2AV87	4HypE	14 ± 0.8	7.8 ± 1	1800
12	Bamb_3769	Q0B950	t3HypD	43 ± 4	13 ± 3	3400
	Bmul_4260	A9AKG8	3HypE	30 ± 1	18 ± 2	1700
	Bmul_4260	A9AKG8	4HypE	1.3 ± 0.04	2.7 ± 0.3	470
16	Csal_2339	Q1QV19	4HypE	0.070 ± 0.005	2.5 ± 0.7	28
17	RSP_3519	Q3IWG2	4HypE	nd ^b	nd ^b	nd ^b
17	Rsph17029_3164	A3PPJ8	4HypE	nd ^b	nd ^b	nd ^b
18	SIAM614_28492	A0NXQ7	4HypE	55 ± 3	3.2 ± 0.5	17000
18	SADFL11_2813	B9R4E3	4HypE	67 ± 5	4.1 ± 0.8	16000
22	Spea_1705	A8H392	t3HypD	0.15 ± 0.03	- ^b	- ^b
22	Swoo_2821	B1KJ76	t3HypD	4.1 ± 0.4	6.7 ± 2	600

a

The reaction is to slow to measure K_m.
b

The reaction is slow to measure kinetic parameters.

Table 3

Growth phenotypes of bacterial strains when grown on the indicated carbon sources

https://doi.org/10.7554/eLife.03275.009

Organism	t4Hyp	c4Hyp	t3Hyp	cis-3-OH-L-proline	L-Pro	D-glucose
Agrobacterium tumefaciens C58	++	++	+	-	+++	+++
Sinorhizobium meliloti 1021	++	++	+	-	+++	+++
Labrenzia aggregate IAM12614	+	+	+	+	+++	+++
Pseudomonas aeruginosa PAO1	++	++	+	-	+++	+++
Paracoccus denitrificans PD1222	+++	+++	+	+	+++	+++
Rhodobacter sphaeroides 2.4.1	+	+	-	-	+++	+++
Rhodobacter sphaeroides 2.4.1?RSP3519	-	+	-	-	+++	+++
Bacillus cereus ATCC14579	++	++	+	+	+++	+++
Roseovarius nubinhibens ISM	++	++	+	-	+++	+++
Escherichia coli MG1655	-	-	-	-	+++	+++
Streptomyces lividans TK24	+++	++	+	ND	+++	+++

‘+++’ represents robust growth (like growth on D-glucose); ++/+ represents slow growth phenotype; ‘--’ represents growth-deficient phenotype; ‘ND’, not determined

Table 4

Transcriptional analysis of PRS members

https://doi.org/10.7554/eLife.03275.010

Organism/Locus Tag	t4Hyp	t3Hyp
*Agrobacterium tumefaciens C58*
A9CKB4	12 ± 2	11 ± 1.5
A9CFV0	3 ± 1	NC
A9CH01	64 ± 5	32 ± 4
*Sinorhizobium meliloti 1021*
Q92WS1	5 ± 1	3 ± 1
Q92WR9	5.5 ± 1.5	3.5 ± 1
*Labrenzia aggregate IAM12614*
A0NXQ7	22 ± 2	5 ± 1
A0NXQ9	12 ± 2	6 ± 2
*Pseudomonas aeruginosa PAO1*
Q9I489	8 ± 2	5 ± 1
Q9I476	35 ± 3	7 ± 2
*Paracoccus denitrificans PD1222*
A1B0W2	2.0 ± 0.5	NC
A1B195	NC	NC
A1B7P4	NC	NC
A1BBM5	4.5 ± 0.5	NC
*Rhodobacter sphaeroides 2.4.1*
Q3IWG2	10 ± 1	NC
*Bacillus cereus ATCC14579*
Q81HB1	4 ± 1	4.5 ± 1
Q81CD7	22 ± 2	18 ± 3
*Roseovarius nubinhibens ISM*
A3SLP2	12 ± 2	4+1.5

Fold change in expression for each gene when grown on the indicated carbon source, relative to growth on D-glucose. The identities of the bacterial species and the protein encoded by each gene are indicated. Fold-changes are the averages of five biological replicates with standard deviation (p value < 0.005). NC, no change.

Table 5

Transcriptional analysis of genome neighborhoods

https://doi.org/10.7554/eLife.03275.011

Organism/Locus tag	UniProt	Enzyme	Cluster	t4Hyp	t3Hyp	L-Pro
Bacillus cereus ATCC 14579
Bc_0905	Q81HB1	ProR	navy	121 ± 11	87 ± 10	NC
Bc_0906	Q81HB0	OCD		20 ± 3	14 ± 2	NC

Bc_2832	Q81CE0	ALDH		630 ± 39	625 ± 57	13 ± 2
Bc_2833	Q81CD9	DHDPS		644 ± 61	498 ± 37	6 ± 0.7
Bc_2834	Q81CD8	ProR	hot pink	594 ± 27	485 ± 29	8 ± 1
Bc_2835	Q81CD7	ProR	teal	408 ± 15	567 ± 33	5 ± 0.5
Bc_2836	Q81CD6	oxidase		623 ± 37	633 ± 42	10 ± 0.6

Streptomyces lividans TK24
SSPG_01342	D6EJL0	DAAO		81 ± 5	20 ± 5	NC
SSPG_01341	D6EJK9	oxidase		65 ± 9	6 ± 0.2	NC
SSPG_01340	D6EJK8	oxidase		225 ± 22	30 ± 3	3 ± 0.4
SSPG_01339	D6EJK7	DHDPS		136 ± 5	16 ± 0.2	NC
SSPG_01338	D6EJK6	ProR	palegreen	171 ± 8	23 ± 1	3 ± 0.2

Agrobacterium tumefaciens C58
Atu_0398	A9CKB4	ProR	orange	14 ± 0.4	16 ± 0.6	NC

Atu_3947	Q7CTP1	DAAO		NC	4 ± 0.2	NC
Atu_3948	Q7CTP2	AlaR		NC	NC	NC
Atu_3949	Q7CTP3	OCD		NC	NC	NC
Atu_3950	Q7CTP4	ALDH		NC	NC	NC
Atu_3951	A9CFU8	LysR		NC	NC	NC
Atu_3952	A9CFU9	DAAO		NC	NC	NC
Atu_3953	Q7CFV0	ProR	blue	NC	NC	NC
Atu_3958	Q7CTQ2	DAAO		NC	NC	NC
Atu_3959	Q7CTQ3	ALDH		NC	NC	NC
Atu_3960	A9CFV4	DHDPS		NC	NC	NC
Atu_3961	Q7CTQ5	GntR		NC	NC	NC
Atu_3985	A9CFW8	ProC		NC	NC	NC

Atu_4675	A9CGZ4	DHDPS		148 ± 2	87 ± 7	NC
Atu_4676	Q7CVK1	MLD2		30 ± 5	40 ± 7	NC
Atu_4678	A9CGZ5	SBP		198 ± 18	79 ± 8	NC
Atu_4682	A9CGZ9	DAAO		294 ± 15	14 ± 3	NC
Atu_4684	A9CH01	ProR	light sky blue	116 ± 14	8 ± 1	NC
Atu_4691	A9CH04	2-Hacid_dh		NC	NC	NC

Fold changes in expression for the indicated gene when grown on the indicated carbon source, relative to growth on Dglucose. Fold changes are the averages of three biological replicates with standard deviation. NC, no change.

Table 6

Data Collection and Refinement Statistics^a

https://doi.org/10.7554/eLife.03275.013

UNIPROT / CLUSTER / PROTEIN	A5VZY6 / 2 / Pput_1285	A5VZY6 / 2 / Pput_1285	Q1QU06 / 2 / Csal_2705	Q8P833 / 2 / XCC_2415	B3D6W2 / 2 / BMULJ_04062	Q4KGU2 / 2 / PFL_1412	Q4KGU2 / 2 / PFL_1412	A6WW16 / 3 / Oant_0439	B9K4G4 / 3 / Avi_7022	B9JQV3 / 9 / Avi_0518
Organism	Pseudomonas putida F1	Pseudomonas putida F1	Chromohalobacter salexigens DSM 3043	Xanthomonas campestris	Burkholderia multivorans	Pseudomonas fluorescens Pf-5	Pseudomonas fluorescens Pf-5	Ochrobacterium anthropi	Agrobacterium vitis S4	Agrobacterium vitis S4
PDBID	4JBD	4JD7	4JCI	4JUU	4K7X	4J9W	4J9X	4K8L	4K7G	4LB0
DIFFRACTION DATA STATISTICS
Space Group	I2	P2₁2₁2₁	P2₁2₁2₁	P2₁2₁2₁	I4₁22	P2₁	P2₁2₁2₁	I222	P4₃2₁2	P4₂2₁2
Unit Cell (Å , °)	a=45.2 b=54.2 c=142.7	a=64.8 b=96.8 c=109.2	a=48.1 b=54.4 c=253.0	a=54.9 b=108.8 c=116.2	a=114.9 b=114.9 c=173.7	a=56.2 b=74.6 c=87.1 β=105.5	a=64.8 b=96.8 c=109.2	a=77.3 b=78.3 c=114.4	a=54.9 b=108.8 c=116.2	a=178.0 b=178.0 c=49.7
Resolution (Å)	1.3 (1.3-1.32)	1.5 (1.5-1.58)	1.7 (1.7-1.79)	1.75 (1.75-1.84)	1.75 (1.75-1.84)	1.6 (.6-1.69)	1.7 (1.7-1.79)	1.9 (1.9-2.0)	2.0 (2.0-2.1)	1.7 (1.7-1.79)
Completeness (%)	99.8 (99.6)	99.5 (98.9)	97.0 (94.0)	99.7 (99.4)	100.0 (100.0)	99.3 (99.5)	99.5 (99.0)	99.8 (100.0)	100 (100)	99.9 (99.9)
Redundancy	3.6 (3.5)	7.3 (7.1)	9.3 (7.8)	7.3 (7.1)	14.3 (13.5)	3.6 (3.5)	6.7 (6.0)	7.2 (7.3)	14.1 (13.2)	10.4 (7.9)
Mean(I)/sd(I)	7.9 (1.4)	18.0 (1.1)	17.5 (3.3)	18.0 (1.1)	14.1 (1.1)	6.9 (1.7)	11.6 (1.5)	6.0 (1.3)	11.6 (3.3)	18.3 (2.7)
R_sym	0.062 (0.735)	0.067 (0.707)	0.073 (0.644)	0.074 (0.725)	0.130 (0.699)	0.093 (0.434)	0.088 (0.531)	0.09 (0.594)	0.17 (0.836)	0.078 (0.745)
REFINEMENT STATISTICS
Resolution (Å)	1.3 (1.3-1.31)	1.5 (1.5-1.52)	1.7 (1.7-1.72)	1.75 (1.75-1.77)	1.75 (1.75-1.78)	1.6 (1.6-1.62)	1.7 (1.7-1.72)	1.9 (1.9-1.97)	2.0 (2.0-2.02)	1.7 (1.72-1.70)
Unique reflections	82749	109888	72128	70700	58574	90740	77405	27674	86628	87548
R_cryst (%)	15.8 (30.4)	15.9 (22.6)	17.1 (23.7)	15.2 (21.5)	13.8 (19.7)	19.7 (28.8)	19.4 (23.5)	16.8 (17.6)	13.6 (19.5)	15.8 (22.9)
R_free (%, 5% of data)	18.4 (31.1)	17.5 (25.4)	20.5 (26.2)	18.4 (26.4)	15.6 (18.5)	23.2 (33.8)	22.5 (27.5)	20.7 (21.7)	16.6 (22.9)	19.2 (27.3)
Residues In Model [Expected]	A1-A308 [1-308]	A(-5)-A308, D(-3)-D308 [1-308]	A(-3)-A169, A171-A309 [1-311]	A(-2)-A312, B(-2)-B312 [1-312]	A(-3)-A310 [1-311]	A1-A310, B1-B310 [1-310]	A1-310, B1-310 [1-310]	A0-A157, A161-A184, A193-A245, A255-280, A289-A332 [1-343]	B5-B342, D(-9)-D342 [1-342]	A1-A323, A326-A344, B0-B346 [1-347]
Residues / Waters / Atoms total	308 / 453 / 3142	626 / 752 / 6225	620 / 494 / 5780	626 / 596 / 5841	314 / 463 / 3223	620 / 537 / 5301	620 / 630 / 5378	305 / 191 / 2824	690 / 780 / 6761	689 / 701 / 6633
Bfactor Protein/Waters/Ligand	17.3 / 31.2 / 21.7	19.3 / 30.5 / 27.9	24.8 / 33.6 / -	23.9 / 35.2 / 37.3	15.6 / 34.0 / 30.6	21.1 / 32.2 / 12.9	22.9 / 34.0 / 16.3	31.3 / 37.7 / -	24.1 / 37.5 / 15.2	25.1 / 36.2 / 17.9
Ligand	Citrate	Sulfate	-	Phosphate / UNL	Phosphate	(PYC) Pyrrole 2-carboxylate	(t4Hyp) Trans- 4OH-L-Proline	-	(PYC) Pyrrole 2-carboxylate	(t4Hyp) Trans- 4OH-L-Proline / Acetate
RMSD Bond Lengths (Å) / Angles (°)	0.008 / 1.283	0.009 / 1.325	0.011 / 1.332	0.010 / 1.26	0.009 / 1.268	0.006 / 1.079	0.006 / 1.093	0.011 / 1.349	0.011 / 1.311	0.010 / 1.320
Ramachandran Favored / Outliers (%)	98.7 / 0.0	96.8 / 0.00	98.2 / 0.00	99.0 / 0.0	97.7 / 0.0	98.7 / 0.0	98.5 / 0.0	98.3 / 0.0	98.0 / 0.3	98.4 / 0.3
Clashscore ^b	2.32 (99^th pctl)	3.02 (98^th pctl)	3.74 (97^th pctl)	4.14 (97^th pctl)	3.12 (97^th pctl)	1.59 (99^th pctl)	1.82 (99^th pctl)	6.6 (93^rd pctl)	2.8 (99^th pctl)	2.2 (99^th pctl)
Overall score^b	1.01 (99^th pctl)	1.29 (95^th pctl)	1.16 (99^th pctl)	1.22 (99^th pctl)	1.16 (99^th pctl)	0.97 (100^th pctl)	0.94 (100^th pctl)	1.36 (98^th pctl)	1.08 (100^th pctl)	1.0 (100^th pctl)

a

Data in parenthesis is for the highest resolution bin
b

Scores are ranked according to structures of similar resolution as formulated in MOLPROBITY

Table 7

Kinetic constants for the proline ketimine reductases (members of the malate/Llactate dehydrogenase 2 [MLD2] and ornithine cyclodeaminase [OCD] superfamilies) that are in the genome neighborhoods of members of the PRS

https://doi.org/10.7554/eLife.03275.015

Cluster	UniProt	Locus tag	Cofactor	k_cat [s^-1]	K_m [mM]	k_cat/K_M[M^-1s^-1]
MLD2_PRS_light skyblue (3)	Q7CVK1	Atu4676	NADPH	32 ± 1	0.33 ± 0.04	99000
MLD2_PRS_light skyblue (3)	Q9I492	PA1252	NADPH	1.6 ± 0.05	0.41 ± 0.06	3900
MLD2_PRS_Red (2)	Q4KGT8	PFL_1416	NADPH	20 ± 0.8	1.1 ± 0.2	18000
	Q0B9S2	Bamb_3547	NADPH	54 ± 13	9.4 ± 4	5700
	A9ALD3	Bmul_4451	NADPH	33 ± 2	7.4 ± 1	4400
MLD2_PRS_indigo (13)	Q4KAT3	PFL_3547^a	NADPH	-	-	2300^b
OCD_PRS_light skyblue (3)	A1B196	Pden_1185	NADPH	260 ± 20	3.1 ± 0.7	85000
	A1B196	Pden_1185	NADH	81 ± 20	16 ± 6	5100
	A3S939	EE36_06353^a	NADPH	6.8 ± 0.7	1.0 ± 0.3	6700
	A3SU01	NAS141_11281^a	NADPH	39 ± 4	1.2 ± 0.4	32000
	A3SU01	NAS141_11281^a	NADH	8.2 ± 4	73 ± 50	110
	Q16D96	RD1_0323^a	NADPH	15 ± 1	0.27 ± 0.07	56000
	Q16D96	RD1_0323^a	NADH	3.7 ± 0.4	11 ± 3	320
	Q5LLV0	SPO3821^a	NADPH	130 ± 20	3.0 ± 0.9	43000
	Q5LLV0	SPO3821^a	NADH	-	-	840^b
	Q3IZJ8	RSP_0854^a	NADPH	66 ± 4	0.43 ± 0.09	150000
	Q3IZJ8	RSP_0854^a	NADH	12^c	-	-
OCD_PRS_navy (5)	Q81HB0	BC_0906	NADPH	15 ± 1	0.47 ± 0.1	31000
	Q81HB0	BC_0906	NADH	19 ± 1	11 ± 2	1800
	Q73CR9	BCE_0995	NADPH	15 ± 1	1.1 ± 0.3	13000
	Q73CR9	BCE_0995	NADH	2.1 ± 0.3	7.6 ± 3	270
	Q6HMS8	BT9727_0800	NADPH	11 ± 1	3.4 ± 0.9	3100
	Q6HMS8	BT9727_0800	NADH	2.1 ± 0.4	18 ± 6	120
	Q63FA5	BCE33L0803	NADPH	5.8^c	-	-
	Q63FA5	BCE33L0803	NADH	0.87 ± 0.1	4.9 ± 2	180
OCD_PRS_olive (12)	Q0B953	Bamb_3766	NADPH	106 ± 4	1.6 ± 0.2	64000
	Q0B953	Bamb_3766	NADH	41 ± 6	7.3 ± 3	5700
	Q2T596	BTH_II1457^a	NADPH	73 ± 2	0.39 ± 0.05	190000
	Q2T596	BTH_II1457^a	NADH	203 ± 23	32 ± 7	6400
	Q3JFG0	BURPS1710b_A2543^a	NADPH	7.8 ± 0.5	0.64 ± 0.1	12000
	Q3JFG0	BURPS1710b_A2543^a	NADH	6.0 ± 1	31 ± 13	190
	A9AKH1	Bmul_4263	NADPH	25 ± 6	4 ± 2	6400
OCD_PRS_blue (1)	Q485R8	CPS_1455	NADPH	35 ± 0.8	1.8 ± 0.2	20000
	Q485R8	CPS_1455	NADH	-	-	170^b
	A3QH73	Shew_2955^a	NADPH	6.7 ± 0.7	1.6 ± 0.6	4300
	A3QH73	Shew_2955^a	NADH	0.37 ± 0.1	26 ± 10	14

a

Highly homologous to MLD2 or OCD which are in the gene context of proline racemase.
b

The enzyme didn’t saturate.
c

K_M is too small (< 0.03mM).

Table 8

Strains used in this study

https://doi.org/10.7554/eLife.03275.020

Organism
Agrobacterium tumefaciens C58
Sinorhizobium meliloti 1021
Labrenzia aggregata IAM12614
Pseudomonas aeruginosa PAO1
Paracoccus denitrificans PD1222
Rhodobacter sphaeroides 2.4.1
Rhodobacter sphaeroides 2.4.1?RSP3519
Bacillus cereus ATCC14579
Roseovarius nubinhibens ISM
Escherichia coli MG1655
Streptomyces lividans TK24

Table 9

Oligonucleotide primers used for construction of the RS3519 knock-out in Rhodobacter sphaeroides 2.4.1

https://doi.org/10.7554/eLife.03275.021

Oligo	Sequence (5'–3')
RS3519F.KO	CATATGATGCGCGTTCAGGACGTGTATAACG
RS3519R.KO	GCTGAGCTCAGAGGACGAGGAAGCCCGCGTCC

Table 10

qRT-PCR primers for transcriptional analysis of individual proline racemase superfamily members

https://doi.org/10.7554/eLife.03275.022

Oligo	Sequence (5'–3')
Atu16s-F	GACACGGCCCAAACTCCTAC
Atu16s-R	GGGCTTCTTCTCCGACTACC
Atu0398-F	TCACCATTGAGAAGGCCAAT
Atu0398-R	GGTTGACGAGGTCCTTCAGA
Atu3953-F	CAGCTTCAGTGGCATCAGG
Atu3953-R	GTGTTGTGCCCAATGATCC
Atu4684-F	GAAGAGGCGCATGAGATTG
Atu4684-R	CGAAACCCAAAGCCTTGTT
Bc16s-F	CTCGTGTCGTGAGATGTTGG
Bc16s-R	TGTGTAGCCCAGGTCATAAGG
Bc0905-F	CTTCGCTGACGGACAAGTAGA
Bc0905-R	TGTACCGCTGTTACGGACAA
Bc2835-F	AACAGACCCGTGTCATCCTG
Bc2835-R	ACTAAGCCAGCCGGTGTATCT
La16s-F	TGGTGGGGTAAAGGCCTAC
La16s-R	TGGCTGATCATCCTCTCAGAC
La28492-F	TGTTGAAGACGAGGCCAAG
La28492-R	AAAAGCCGAGCTGTTCGTT
La28502-F	CGCGTAATCGACAGCCATA
La28502-R	GGCACAGAAATCGAGATGCT
Rs16s-F	ACACTGGGACTGAGACACGG
Rs16s-R	TACACTCGGAATTCCACTCA
Rs3519-F	AGGACATCGCCTTCGAACT
Rs3519-R	CGATGATGCCGAAATAGTTG
Pa16s-F	TCACACTGGAACTGAGACACG
Pa16s-R	ATCAGGCTTTCGCCCATT
Pa1255-F	CCACCCTCTGGGAACAGTC
Pa1255-R	TCGTTGAGGACGAAGTTGC
Pa1268-F	AACAGTGGCTACCTCGGCA
Pa1268-R	TCGCCGACCGGTGTCTCGAT
Rn16s-F	ATCTGTGTGGGCGCGATT
Rn16s-R	GTGAGCGCATTGGTGGTCT
Rn08250-F	TATGGCGGCGACAGTTTC
Rn08250-R	GACGGCTCGAGCGTAAAC
Pd16s-F	GACTGAGACACGGCCCAGA
Pd16s-R	TCACCTCTACACTCGGAAT
Pd1045-F	TCGGACTACTATGTGCCGATG
Pd1045-R	CCTGATCGAGGCCAAAGAC
Pd1184-F	GCAATTTCGTGTTGAACGAG
Pd1184-R	CATGATGATCCAGCCCATCT
Pd3467-F	CTTCGCAGCCCTGTTCAT
Pd3467-R	GACCAGCCCTTCCTCGAT
Pd4859-F	GGCAAGGTGGACATCGAATA
Pd4859-R	CCTCGGGGTAAAGGAAGC
Sm16s-F	CGTGGGGAGCAAACAGGATT
Sm16s-R	CTAAGGGCGAGGGTTGCGCTC
Sm20268-F	CTGGCAAGGTGGACATCAC
Sm20268-R	GTAAGGCGCACTTCCTCAA
Sm20270-F	CGCCATGTCAATCTCCTGGT
Sm20270-R	GGCAGCATCCACGATCACGA

Table 11

qRT-PCR primers for transcriptional analysis of genome neighborhoods

https://doi.org/10.7554/eLife.03275.023

Primer	Sequence (5'–3')
Sliv-Sco16srRNA-F	CCGTACAATGAGCTGCGATA
Sliv-Sco16srRNA-R	GAACTGAGACCGGCTTTTTG
Sliv-Sco6289-F	GACCCTGAAGGTCGTCGTC
Sliv-Sco6289-R	GGTGACCGTGACGTCCAT
Sliv-Sco6290-F	GTCTTCTGCGGCATCGG
Sliv-Sco6290-R	AGTCATCGTCGTCCTCCA
Sliv-Sco6291-F	GCCGACCTCGACGAAGA
Sliv-Sco6291-R	TTGTCGGTTTCACTGCTGTC
Sliv-Sco6292-F	CATCGACACCAAGGTGGAC
Sliv-Sco6292-R	TGACCCCGACGATGTACC
Sliv-Sco6293-F	GACTACGGCGTGCTCTTCAT
Sliv-Sco6293-R	CTCGGTGACCTCGACCAT
Bc0905-F	CTTCGCTGACGGACAAGTAGA
Bc0905-R	TGTACCGCTGTTACGGACAA
Bc0906-F	ACTACGAACGCAACCACACC
Bc0906-R	CGGAACTTGAAGGTCTCCTGT
Bc2832-F	TACCAGGCTTTGGTCCTGAA
Bc2832-R	ATTTGCCGCCAAGCTCTAAC
Bc2833-F	GGATGGGTTTCAGTAGCAGGA
Bc2833-R	CCTAGTCTTGGATAGCGAGAAGG
Bc2834-F	AGGTGCGTATTCGCCAGAAA
Bc2834-R	CCTGGCGAACGTACGATAAA
Bc2835-F	AACAGACCCGTGTCATCCTG
Bc2835-R	ACTAAGCCAGCCGGTGTATCT
Bc2836-F	CCTTGCATTCTCGCTTCTGT
Bc2836-R	AATCTTAGGAGCCCACACACC
Atu3947-F	TCCGGCCAAGTATGTGAAAG
Atu3947-R	CTATAGCCGTTCGCAGCAAG
Atu3948-F	ATTTCGCCCGTGATCTGTC
Atu3948-R	CGGCATCCACAATAATCCAG
Atu3949-F	GCGAACAGGCTGAAGAGATG
Atu3949-R	CGGCGGTAATTCCTGTTTG
Atu3950-F	GCTGCCGAACATATCAAGGT
Atu3950-R	GACCTTCGCGGTTATCTGGT
Atu3951-F	TGACGGACTCCAGCCTTATC
Atu3951-R	ATGTAACATCGGCGTGGTCT
Atu3952-F	GATATCGTCAAGGGCGGTTT
Atu3952-R	ACGCAGAGCCTTCATGTGTT
Atu3953-F	CAACGTCGCCAGTTACCTTC
Atu3953-R	GGCTGAGATCAACGACATCC
Atu3958-F	GGCGGCTGATACACATCTTC
Atu3958-R	AAAGTTGGTGCTTCGTCAGG
Atu3959-F	CATTCCTGACACGATCCACA
Atu3959-R	CAGCATCAGCAAAGGGAAGT
Atu3960-F	GAATGTCGTCGCCATCAAG
Atu3960-R	TCGTAGAGTGCCACATGCTC
Atu3961-F	TTCGGCACTTCTTTCTGGTC
Atu3961-R	GCTCGCCTGCAGATAAACA
Atu4675-F	TTCCTGTTATCGTCGGCACT
Atu4675-R	GCCTTGAAGTGAGCCTTCTG
Atu4676-F	ACGGCTATCGTGAAGGTCAA
Atu4676-R	GAATAGCTCGGGCACATCAC
Atu4682-F	TCCTCAGAAAGACCGACACC
Atu4682-R	GTGAATGTGCCGCAGGTAA
Atu4684-F	CCTCGGCAAACTCAAGGTC
Atu4684-R	GCGAAGAGGCAGAAGGAAA
Atu4691-F	AAGGGCGATATGGGTCTTTC
Atu4691-R	GAGCTCTTCGATGCTGTCGT

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Suwen Zhao
Ayano Sakai
Xinshuai Zhang
Matthew W Vetting
Ritesh Kumar
Brandan Hillerich
Brian San Francisco
Jose Solbiati
Adam Steves
Shoshana Brown
Eyal Akiva
Alan Barber
Ronald D Seidel
Patricia C Babbitt
Steven C Almo
John A Gerlt
Matthew P Jacobson

(2014)

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

eLife 3:e03275.

https://doi.org/10.7554/eLife.03275

Figures

The reactions catalyzed by proline racemase (ProR), 4R-hydroxyproline 2-epimerase (4HypE), and trans-3-hydroxy-L-proline dehydratase (t3HypD) and the metabolic pathways in which they participate.

Sequence similarity networks (SSNs) for the PRS.

The genome neighborhood network (GGN) for the PRS.

Library of proline and proline betaine derivatives tested for ESI-MS screening.

Structures of members of the PRS.

Sequence divergent members of the ornithine cyclodeaminase superfamily (OCDS) have been the assigned novel pyrroline-2-carboxylate reductase (Pyr2C reductase) function in this work.

Mapping members of GNN clusters back to the SSN for the PRS.

Experimentally characterized enzymes reported by Swiss-Prot (small colored circles) and newly characterized in this work (large colored circles).

Demonstration of the 4HypE, 3HypE, and t3HypD reactions by ¹H NMR.

Representative ¹H NMR spectra for ?¹-pyrroline-2-carboxylate (?¹-Pyr2C) reductase activity.

Tables

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The reactions catalyzed by proline racemase (ProR), 4R-hydroxyproline 2-epimerase (4HypE), and trans-3-hydroxy-L-proline dehydratase (t3HypD) and the metabolic pathways in which they participate.

Sequence similarity networks (SSNs) for the PRS.

The genome neighborhood network (GGN) for the PRS.

Library of proline and proline betaine derivatives tested for ESI-MS screening.

Structures of members of the PRS.

Sequence divergent members of the ornithine cyclodeaminase superfamily (OCDS) have been the assigned novel pyrroline-2-carboxylate reductase (Pyr2C reductase) function in this work.

Mapping members of GNN clusters back to the SSN for the PRS.

Experimentally characterized enzymes reported by Swiss-Prot (small colored circles) and newly characterized in this work (large colored circles).

Demonstration of the 4HypE, 3HypE, and t3HypD reactions by 1H NMR.

Representative 1H NMR spectra for ?1-pyrroline-2-carboxylate (?1-Pyr2C) reductase activity.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Demonstration of the 4HypE, 3HypE, and t3HypD reactions by ¹H NMR.

Representative ¹H NMR spectra for ?¹-pyrroline-2-carboxylate (?¹-Pyr2C) reductase activity.