65
Chapter 3: Results and Discussion
Samples collected in two years (as described in Methods -section "fruiting bodies") from different parts of Italy were used for proteomic analysis. The degree of maturation of fruiting bodies was measured according to (Zeppa et al., 2002) and reported as stage 5, with the presence of 80-100% mature spores that were yellow-reddish brown in color with reticulate ornamentation (Garnero and Bonfante 2000).
66 The protein extraction protocol was set-up in order to increase the yield and quality of the protein extracted. To reach this goal, the Urea molarity was increased to 8 M, to increase the number of detectable spots. Two washing steps (acetone 80%) were added to remove polyphenols and other contaminants from the protein extract.
At least three independent replicate gels were performed for each year of sample collected. Each gel was a result of a separate extraction to minimize the experimental error.
Gel images were scanned and their images were used for the bioinformatic analysis, performed on the base of the number, the size and intensity of the spots.
Different bioinformatic analysis were performed. The first analysis was carried out on the base of the samples collected in two years from different regions (Tuscany, Umbria, Marche, Piedmont) whereas the other analysis included samples collected in three and four years and host plant comparisons to verify the effect of different host plants in samples collected from the same geographical area.
3.1 Analysis of samples collected in two years
Bionformatic analyses were initially performed on samples collected in two years.
Gel images were loaded, checked for any saturation problems and analyzed using a San Miniato gel image as internal standard. The comparison of protein patterns in different accessions of Tuber revealed the presence of quantitative differences (relative abundance) that remained constant (Fig 31, Fig 32) as well as the number of the spots in the gels.
67 After bioinformatic analysis, 60 spots showed statistically significant differences in Opitcal Density (OD) values (which in turn indicate protein expression levels) that potentially could provide a basis to distinguish between samples. Among these, seventeen were most suitable to describe the area of origin, as assessed by principal component analysis (PCA) and were therefore selected for MS analysis (Fig 31, Fig 32, and Fig 33). The PCA results (Fig 32), showed that it is possible to distinguish between samples from different regions (Tuscany, Piedmont, Marche, Umbria) by examining on the expression level of these 17 spots.
Figure 31: Representative 2D gel from the San Miniato sample (1 mg of protein extract)
stained with colloidal Coomassie. Spots selected by bioinformatic analysis to discriminate between different areas of origin as shown here.
68
Figure 32: Principal Component Analysis obtained on the base of the ability of the 17 spots
to discriminate the area of origin. Pink circles represent Tuscany’s samples; Green olive circles represent Umbria samples; Yellow circles represent Marche samples; Blue circles represent Piedmont samples
For instance spots 2 and 8 strongly contribute to form a cluster where the sample Umbria shows average normalized intensity values lower than the other accession areas (Fig 33), while the spots 6 and 7 were able to distinguish samples coming from Piedmont (Alba 1 and 2) from all the others. The sample collected in the Marche region on the other hand, may be identified by spot 12, while the accessions from Tuscany (the largest group), showed consistently differences for spots 3 and 5 (Fig 33). Furthermore, a post-analysis test for the ANOVA results comparing pairs of samples (Fig 33, Fig 34) showed that some samples are differentiable within the area of origin (namely Tuscany). For example, the sample Crete Senesi, presents the differences of expression in spots 1 and 3, while spots 4 and 10 distinguish the sample Lucca from the others.
69
70
Figure 33b: Intensity levels of the spots selected for MS analysis. Enlarged areas from 2D-E
representative gels are shown and the relative quantitation (n= 6) are reported as values expressed in log10 normalized volume (spot optical density) with ± SE. *= p value < 0,05,
71 Mass spectrometry analysis allowed to get a protein sequence for 15 out of 17 spots. The protein identification was performed using the whole Tuber spp. database, mainly composed of proteins of T. melanosporum, cause the lack of a specific protein dataset for T. magnatum. Two spots (10 and 12) failed to give protein identifications while spots 5, 13, 16 and 17 gave rise to the identification of two different proteins in each spot. 17 different proteins were identified (16 through ESI-TOF analysis and 1 by MALDI analysis) even though in spots 2 and 8, and 6 and 9 different isoform of the same proteins were identified (Table 5, 6).
Table 5: Proteins identified by ESI-Quad TOF analysis
Spot No. (a) Acc. No. (b) Protein Description Organism Score (c) Sequence Coverage % (d) Mw kDa Obs/ Theo (e) pI Obs/Theo (f) Identified peptides (MS/MS) (g) Tuber Gene annotation (h) Protein Blast relation (i) 1 D5G797 Whole genome shotgun sequence assembly, scaffold_131, strain Mel28 T. melanosporum (Perigord truffle) 104 4 78 46,2 6,2 5,82 2 GSTUM_00002523001 A 2 D5GJY5 Whole genome shotgun sequence assembly, scaffold_55, strain Mel28 T. melanosporum (Perigord truffle) 378 11 162 86,3 5,4 5,16 7 GSTUM_00009270001 B 3 D5GGN7 Dihydrolipoyl dehydrogenase T. melanosporum (Perigord truffle) 137 6 81 54 5,7 6,72 3 GSTUM_00007439001 4 D5G8F0 Whole genome shotgun sequence assembly, scaffold_15, strain Mel28 T. melanosporum (Perigord truffle) 164 28 22,5 20 5,7 5,41 4 GSTUM_00004791001 C 5 D5GJ78 S-adenosylmethionine synthase T. melanosporum (Perigord truffle) 871 47 57 41,8 5,8 5,7 15 GSTUM_00008874001 D5G9M7 Whole genome shotgun sequence assembly, scaffold_169, strain Mel28 T. melanosporum (Perigord truffle) 114 12 57 57,8 5,8 9,93 5 GSTUM_00003332001 D 6 D5GNA2 Whole genome shotgun sequence assembly, scaffold_8, strain Mel28 T. melanosporum (Perigord truffle) 125 22 22 22 5,5 6,21 4 GSTUM_00011192001 E 7 D5GAF9 Whole genome shotgun sequence assembly, scaffold_18, strain Mel28 T. melanosporum (Perigord truffle) 146 8 45 33 5,1 6,02 4 GSTUM_00005271001 F
72 Spot No. (a) Acc. No. (b) Protein Description Organism Score (c) Sequence Coverage % (d) Mw kDa Obs/ Theo (e) pI Obs/Theo (f) Identified peptides (MS/MS) (g) Tuber Gene annotation (h) Protein Blast relation (i) 8 D5GJY5 Whole genome shotgun sequence assembly, scaffold_55, strain Mel28 T. melanosporum (Perigord truffle) 170 9 91 86,3 5,4 5,16 6 GSTUM_00009270001 9 D5GNA2 Whole genome shotgun sequence assembly, scaffold_8, strain Mel28 T. melanosporum (Perigord truffle) 1206 35 25 22 6,1 6,21 6 GSTUM_00011192001 G 11 D5G6L6 Whole genome shotgun sequence assembly, scaffold_121, strain Mel28 T. melanosporum (Perigord truffle) 118 21 41 31,3 5,2 6,01 5 GSTUM_00002097001 13 D5GC43 Whole genome shotgun sequence assembly, scaffold_201, strain Mel28 T. melanosporum (Perigord truffle) 149 18 38 33,5 5.1 5,06 5 GSTUM_00000555001 H
D5GAC6 Probable Xaa-Pro
aminopeptidase P T. melanosporum (Perigord truffle) 131 5 38 69,2 5.1 5,28 3 GSTUM_00005237001 14 D5GCN2 Whole genome shotgun sequence assembly, scaffold_218, strain Mel28 T. melanosporum (Perigord truffle) 83 22 19 20,7 5.4 8,64 3 GSTUM_00000743001 I 15 D5G620 dehydrogenase Malate T. melanosporum (Perigord truffle) 115 8 18 36,7 5.5 8,79 3 GSTUM_00001731001 16 D5GE86 Whole genome shotgun sequence assembly, scaffold_26, strain Mel28 T. melanosporum (Perigord truffle) 105 14 28,2 26,8 5.5 7,1 4 GSTUM_00006427001 L 17 D5G5R4 Whole genome shotgun sequence assembly, scaffold_112, strain Mel28 T. melanosporum (Perigord truffle) 376 11 45,6 46,9 5.7 5,97 4 GSTUM_00001447001 M Q1ACW3 NADP-dependent mannitol dehydrogenase Tuber borchii 314 26 45,6 38 5.7 5,69 7 DQ223686.1
Table 5: List of proteins identified by ESI-Quad TOF analysis. (a) as indicated in Figure 31; (b) UniProtKB accession number; (c) Mascot score; (d) percent sequence coverage; (e) gel-observed vs. theoretical molecular weights * Observed and theoretical weight may differ on the base of the real molecular weight of the protein in T. magnatum; (f) observed vs. theoretical isoelectric points; (g) number of matched peptides; (h) gene reference code; (i) numerical correlation to proteins obtained by blast analysis (Table 7). spot 10 and spot 12, no protein identification
73
Table 6: Proteins identified by MALDI-TOF MS analysis
Spot No. (a)
Acc. no.
(b) Protein name Organism
Mw kDa Obs/ Theo (c) pI Obs/Theo (d) Score (e) Seq. cov (f) Matc.
pep. (g) Biol. Proc. (h)
16 D5GJE0 hypothetical protein T. melanosporum Mel28 28.2 25.6 5.5 5.77 80 40% 11
Similar to: mitochondrial peroxiredoxin PRX1 UniProtKB
Acc. no. (A7EHB2)
Table 6: List of proteins identified by MALDI-TOF peptide mass fingerprint analysis. (a) as indicated in Figure 31; (b) UniProtKB accession number; (c) gel-observed vs. theoretical molecular weights; (d) observed vs. theoretical isoelectric points; (e) Mascot score; (f) percentage sequence coverage; (g) number of matched peptides; (h) biological process obtained after protein blast analysis.
All of the proteins, with the exception of Q1ACW3 (NADP-dependent mannitol dehydrogenase) had been previously found in T.melanosporum (Table 5, 6). To ensure a putative functional identification, protein blast (UniprotKB) was performed when no other information was available (results reported in Table 7).
Some of the proteins identified can be grouped according to the metabolic pathways that they belong to. Spots 4 and 5 belong to the methionine metabolism. Spot 5 (Table 5) is an S-adenosylmethionine synthetase; this enzyme, similarly to the peptide methionine sulfoxide reductase (spot 4, Table 7), could function protecting cell against oxidative damage (Weissbach and Etienne 2002). S-adenosylmethionine synthase appears to take part in cystein/methionine biosynthesis and interconversion, playing a key role in the production of hydrogen sulfide (Martin et al., 2010). Hydrogen sulfide is a precursor of many volatile compounds, two of
74 which, dimethyl trisulfide and dimethyl disulfide (Landaud et al., 2008), are among the main volatile compounds responsible for the T. magnatum flavor.
75
Figure 34b: Tukey Post Test results performed on the base of the ANOVA results, for the
comparison of samples’s pairs. *= p value < 0,05, ** = p-value < 0,01, *** = p-value < 0,001,
**** = p-value < 0,0001; Sm=San Miniato, A1=Alba 1, A2=Alba 2, Mo=Montaione,
M=Mugello, S=Crete Senesi, L=Lucca, Msa= Marche, Um=Umbria, Cas P=Casentino
76 Dihydrolipoyl dehydrogenase (spot 3), plays a role in cell redox homeostasis, while glyoxal oxidase (spot 2 and 8) catalyzes the oxidation of several aldehydes producing extracellular H2O2 (Kersten 1990). It is
interesting to observe that glyoxal oxidase shares some traits with another fungal enzyme, galactose oxidase. The critical active site residues typical of radical copper oxidases are conserved between these two enzymes (Whittaker and Kersten 1999). Galactose oxidase catalyzes the oxidation of primary alcohols to aldehydes and is reported to be a monomeric enzyme of 68.5 kDa (Jazdzewski and Tolman 2000), though previously was considered a dimer or higher polymer (Hamilton et al., 1978). In our work glyoxal oxidase has been identified at two different molecular weights (91 and 162 kDa, referring to spot 2 and 8), although at the same pI suggesting that spot 2 protein might be a dimer of glioxal oxidase. Also the intensity values are similar for both spots and correlate well with transcript data, as demonstrated by Alba 1 and Umbria samples that represent respectively the highest and lowest expression level.
Table 7: Additional information on the proteins obtained by blast analysis
Spot No. (a) Protein Blast relation (b) Acc. No. (c)
Protein Name Organism Protein
Length (d)
E-Value (e) 1 A G2YT91 Similar to peptidase (Secreted
protein)
Botryotinia
fuckeliana 383 1.0×10-120
2, 8 B B2WA62 Glyoxal oxidase Pyrenophora
tritici-repentis 825 0.0
4 C B2WK75 Peptide methionine sulfoxide
reductase msrB/msrA
Pyrenophora
tritici-repentis 184 3.0×10-96
5 D Q4WLU4 RNP domain protein Neosartorya
fumigata 480 1.0×10-120
6, 9 E C4JW16 NAD(P)H:quinone oxidoreductase,
type IV Uncinocarpus reesii 203 1.0×10-100
7 F E5ABQ9 Similar to pyridoxine biosynthesis protein
Leptosphaeria
77 Spot No. (a) Protein Blast relation (b) Acc. No. (c)
Protein Name Organism Protein
Length (d)
E-Value (e) 11 G C5FLT9 3-oxoacyl-[acyl-carrier-protein]
reductase Arthroderma otae 289 2.0×10-92
13 H A4D9A0 BAR protein Neosartorya
fumigata 305 1.0×10-137
14 I B8MCW9 AhpC/TSA family protein Talaromyces
stipitatus 181 3.0×10-50
16 L C1G7T9 HD domain-containing protein Paracoccidioides
brasiliensis 224 4.0×10-62
17 M G0RU75
Fructose bisphosphate aldolase Hypocrea jecorina 360 0.0
Table 7: Protein Blast results of the identified proteins mentioned in Table 5. (a) as indicated in Figure 31; (b) alphabetical correlation to proteins previously reported (Table 5); (c) UniProtKB, accession number; (d) as reported from blast output; (e) Expect value, the lower the E-value the higher is the “significance” of the match
In order to obtain further data in addition to protein identification, we performed a gene expression analysis through qPCR on selected genes. Two technical replicates were performed for each qPCR analysis. When it was not possible to design specific primers (e.g. malate dehydrogenase) due to lack of information, no data were obtained. qPCR was also useful in the case of double protein identification, to try to determine which of the two is responsible for the expression changes in the protein spots observed between truffles of different origin. 10 out of 17 genes related to as many proteins were analyzed by this method. For the remaining 7 genes, it proved impossible to select an efficient set of primer due to the current lack of sequence information. The genes analyzed in the Lucca sample present low levels of expression when compared to all other samples, probably due to the quality of the RNA that was lower than in others. With the exception of this sample, the gene expression data generally correlated well with the related protein levels, as shown by spot 2,8 and 7 (Fig 33, Fig 35). In spot 2,
78 both the protein levels of D5GJY5 (putative glyoxal oxidase, Table 7) and the corresponding transcripts were highest in the Crete Senesi sample.
Figure 35: Coordinate regulation of gene related proteins. The Lucca sample was selected
as internal control and it represents the less expressed sample. (n=4), Cas P=Casentino
The same was true for spot 7 (D5GAF9, putative pyridoxine biosynthesis protein, Table 5), in which both protein and transcript levels were highest in the San Miniato sample. In four cases (spots 5, 13, 16 and 17) two proteins were identified for each spot. For these, both relative transcripts were analyzed, except for spot 16. As shown in Fig 35, transcript levels for protein D5G9M7 fit better with the trend observed for the protein spot 5, rather than transcriptional level of D5GJ78. Similarly in spot 13, the transcript level related to protein D5GAC6 is well correlated with the 2D spot trend. This may suggest that those proteins are the ones that most contribute to the difference of intensity detected in spot 5 and 13. Transcript level for protein D5G5R4 (spot 17) well correlates with its protein level; but this does not occur for protein Q1ACV3.
79
3.1.1 Summary list of the identified proteins and their biological relevance (Table 5, 6, 7)
Malate dehydrogenase EC 1.1.1.37
MDH was inferred from T. melanosporum. It is an enzyme of the TCA cycle that catalyzes the interconversion of malate into oxalacetate linked to the oxidation/reduction of dinucleotides coenzymes. There is also a MDH in the cytosol; using the malate-aspartate shuttle the malate can pass the mitochondrial membrane and get transformed to OAA. It is found in all eucariotic cells as the two enzymes. Oxaloacetate plays a crucial role in many metabolic pathways including TCA cycle, glyoxylate cycle, gluconeogenesis, facilitating the exchange between cytoplasm and subcellular organelles. Consequently MDH has been isolated from many sources including eubacteria, archaea, fungi, plants, animals and from various organelles such as mitochondria, chloroplasts, glyoxysomes and peroxisomes (Goward and Nicholls 2008).
NADP-dependent mannitol dehydrogenase EC 1.1.1.138
NADP-dependent mannitol dehydrogenase was inferred from T.borchii, and T. melanosporum. Mannitol, a six-carbon non-cyclic polyol, is the most abundant sugar-alcohol and it is present in all organisms. It is associated with fungal species because fungi are major producers and accumulators of this compound. Mannitol can function as reserve carbon source, osmoprotection, translocation of carbohydrates in the mycorrhiza, fungal-plant interactions (quencher of ROS) promoting pathogen colonization. There is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defences.
80 Mannitol dehydrogenase catalyzes the direct conversion of mannitol to mannose and is a key regulator of mannitol pool size. Fungi normally produce mannitol. As Ceccaroli et al., (2007) state, the fungal-plant symbiosis leads to the formation of ectomycorrhiza which allows metabolic exchanges: at the symbiotic interface the fungal and root cortical cells compete for monosaccharides, such as glucose and fructose, generated from plant-derived sucrose. The monosaccharides are then quickly converted into specific polyols: in the case of Tuber borchii they are mainly metabolized to mannitol as storage molecule. Mannitol is present during active growth of mycelium and it is continuously synthesized and metabolized in the hyphae by the mannitol cycle. In this pathway the mannitol dehydrogenase catalyzes the reversible conversion of fructose to mannitol.
In an old study carried out by Bonnet (1959) ninety-one species of basidiomycetes were examined: mannitol and threalose were present in 86 and 82 species respectively and in no case were both absent. Other soluble carbohydrates are generally much less common and abundant in fungi.
Fructose bisphosphate aldolase EC 5.2.13
Fructose bisphosphate aldolase was inferred from T. melanosporum. This enzyme catalyzes one of the aldol reactions. The substrate fructose 1,6-bisphosphate (F1,6BP) is broken down into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). It takes part in the glycolysis pathway.
81
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) EC 1.2.1.12
GAPDH was inferred from T. borchii and T. melanosporum. It catalyses the conversion of glyceraldehyde 3-phosphate to glycerate 1,3-bisphosphate. This metabolic function has been long established. Recently has been implicated in non-metabolic process like transcription activation, initiation of apoptosis and ER to Golgi vesicle shutting.
Broetto et al., (2010) detected the GAPDH protein in the conidial cell wall of the entomopathogenic fungus Metarhizium anisopliae with some possible implications for the host interaction process, possibly with adhesion activity as reported for other pathogens.
Peroxiredoxin EC 1.11.1.15
Peroxiredoxin protein was inferred from T. melanosporum. Peroxiredoxins are a ubiquitous family of antioxidant enzymes; their physiological importance is illustrated by their relative abundance. It has been considered (Wood et al., 2003) an important emerging family of sulfhydryl-linked (redox-active cysteine) antioxidant proteins, apparently ubiquitously present in all organisms. Mitochondria are the major intracellular sites of oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. Cao et al., (2007) conclude that mitochondrial peroxiredoxin form a vital link in an integrated cellular antioxidant defense network that minimizes ROS-mediated damage and ensures that cells mount appropriate responses to increased levels of oxidative stress via the upregulation of key cell signaling pathways.
82
Glyoxal oxidase EC
1.1.3.-Glyoxal oxidase was inferred from T.melanosporum. The enzyme catalyzes the oxidation of aldehydes, producing H2O2. Lignin degradation performed
by peroxidases (ligninases) requires extracellular H2O2. These extracellular
enzymes oxidize and partially depolymerize lignin. Glyoxal oxidase is an extracellular protein that is necessary for the oxidation activity of ligninolyticperoxidases in Phanerochaete chrysosporium (Kersten et al., 1995; Cullen 1997). The ability to degrade lignin might be a necessary step for Tuber infection. Glyoxal oxidase is also expressed by the pathogenic fungi Fusarium oxysporum and Trichoderma atroviride. (Menotta et al., 2004)
Dihydrolipoyl dehydrogenase EC 1.8.1.4
Dihydrolipoyl dehydrogenase was inferred from T.melanosporum. It is a component of the multienzyme 2-oxo-acid dehydrogenase complexes, such as the pyruvate dehydrogenase complex, responsible for the conversion of pyruvate to acetyl-CoA (Mattevi et al., 1992). It was first shown to catalyse the oxidation of NADH by methylene blue; this activity was called diaphorase.
The term ‘‘diaphorase’’ refers to a ubiquitous class of flavin-bound enzymes that show NADH dehydrogenase activity, where the electron acceptor is generically an oxidized synthetic dye such as methylene blue or 2,6- dichlorophenolindophenol (DCPIP) (Chahraborty et al., 2008).
Peptide methionine sulfoxide reductase EC 1.8.4.11
Peptide methionine sulfoxide reductase was inferred from T.melanosporum. One of the amino acids most easily oxidized in proteins is methionine, which is converted to methionine sulfoxide. This enzyme (also known as
83 MsrA) catalyzes the reduction of methionine sulfoxide in proteins back to methionine, protecting cells against oxidative damage due to condition of oxidative stress. (Weissbach et al., 2002). MsrA may not only function to restore biological activity to proteins inactivated by Met oxidation (for review see Ref. 6), but the reversible oxidation/ reduction of Met in proteins may be one of the prime mechanisms that cells use to scavenge ROS (reactive oxygen species) and RNI (nitrogen intermediates) before they damage cellular constituents (Weissbach et al., 2002).
S-adenosylmethionine synthase EC 2.5.1.6
S-adenosylmethionine synthetase was inferred from T.melanosporum. It is the enzyme that catalyzes the formation of S-adenosylmethionine (AdoMet) from methionine and ATP (Horikawa et al., 1990). AdoMet is an important methyl donor for transmethylation and is also the propylamino donor in polyamine biosynthesis. Almost all organisms have this functional enzyme.
In bacteria there is a single isoform of AdoMet synthetase (gene metK), there are two in budding yeast (genes SAM1 and SAM2) and in mammals while in plants there is generally a multigene family. The sequence of AdoMet synthetase is highly conserved throughout isozymes and species. The active sites of both the Escherichia coli and rat liver MAT reside between two subunits, with contributions from side chains of residues from both subunits, resulting in a dimer as the minimal catalytic entity. The side chains that contribute to the ligand binding sites are conserved between the two proteins. S-Adenosylmethionine-mediated methylation of DNA is known to have regulatory effects on DNA transcription and chromosome structure.
84
RNP domain-containing protein
RNA binding protein (RBPs) was inferred from T.melanosporum. This protein shown to function as central regulators in the post-transcriptional regulation of RNA metabolism during diverse cellular processes, including growth, development, and stress responses. Typical RBPs contain one or more RNA recognition motifs (RRM, also known as RBD or RNP domain) (Kang et al., 2012).The RNP (ribonucleo protein) domain is found in a number of proteins involved in processing and transport of mRNA precursors. The RNP motif is the most common RNA-binding motif and is found in over 200 distinct RNA-binding proteins. Some proteins in the RNP family are involved in the selection of alternative splice sites and RNA metabolism, and play essential roles in development.
NADH-quinone oxidoreductase EC 1.6.99.3
NADH-ubiquinone oxidoreductase was inferred from T.melanosporum. It is the first of three multisubunit enzyme complexes in the inner membranes of mitochondria forming the electron transport chain from NADH to oxygen. It is one of the most complicated enzyme complexes known, containing one noncovalently bound flavin mononucleotide and at least five iron–sulphur clusters recognized by their electron paramagnetic resonance signals (Schuler et al., 1999).
Pyridoxine biosynthesis protein
Pyridoxine biosynthesis protein was inferred from T. melanosporum. It is an enzyme required for the production of pyridoxine (Vitamine B6) and it may
85 be involved in growth arrest and cellular response to nutrient limitation. Required for growth in the presence of low level of intracellular pyridoxine (Rodríguez-Navarro et al., 2002, Stolz and Vielreicher 2003).
3-oxoacyl-[acyl-carrier-protein] reductase EC 1.1.1.100
3-oxoacyl-[ACP] reductase was inferred from T.melanosporum. It also called 3-ketoacyl-acyl carrier protein reductase, is an enzyme of fatty acid biosynthesis found in many plant and bacterial species; it belongs to the short-chain dehydrogenases/reductases (SDR) family (Tan K-C., et al., 2008). This enzyme is involved in type II fatty acid biosynthesis, where the individual metabolic transformations are carried out by different enzymes rather than by a single enzyme as occurs in type I fatty acid biosynthesis (Campbell and Cronan 2001).
