An Integrated Approach to Uncover Driver Genes in Breast Cancer Methylation Genomes

Background

Cancer cells typically exhibit large-scale aberrant methylation of gene promoters. Some of the genes with promoter methylation alterations play “driver” roles in tumorigenesis, whereas others are only “passengers”.

Results

Based on the assumption that promoter methylation alteration of a driver gene may lead to expression alternation of a set of genes associated with cancer pathways, we developed a computational framework for integrating promoter methylation and gene expression data to identify driver methylation aberrations of cancer. Applying this approach to breast cancer data, we identified many novel cancer driver genes and found that some of the identified driver genes were subtype-specific for basal-like, luminal-A and HER2+ subtypes of breast cancer.

Conclusion

The proposed framework proved effective in identifying cancer driver genes from genome-wide gene methylation and expression data of cancer. These results may provide new molecular targets for potential targeted and selective epigenetic therapy.     

Short-Term Soy Isoflavone Intervention in Patients with Localized Prostate Cancer: A Randomized,Double-Blind,Placebo-Controlled Trial

Purpose

We describe the effects of soy isoflavone consumption on prostate specific antigen (PSA), hormone levels, total cholesterol, and apoptosis in men with localized prostate cancer.

Methodology/Principal Findings

We conducted a double-blinded, randomized, placebo-controlled trial to examine the effect of soy isoflavone capsules (80 mg/d of total isoflavones, 51 mg/d aglucon units) on serum and tissue biomarkers in patients with localized prostate cancer. Eighty-six men were randomized to treatment with isoflavones (n = 42) or placebo (n = 44) for up to six weeks prior to scheduled prostatectomy. We performed microarray analysis using a targeted cell cycle regulation and apoptosis gene chip (GEArrayTM). Changes in serum total testosterone, free testosterone, total estrogen, estradiol, PSA, and total cholesterol were analyzed at baseline, mid-point, and at the time of radical prostatectomy. In this preliminary analysis, 12 genes involved in cell cycle control and 9 genes involved in apoptosis were down-regulated in the treatment tumor tissues versus the placebo control. Changes in serum total testosterone, free testosterone, total estrogen, estradiol, PSA, and total cholesterol in the isoflavone-treated group compared to men receiving placebo were not statistically significant.

Conclusions/Significance

These data suggest that short-term intake of soy isoflavones did not affect serum hormone levels, total cholesterol, or PSA.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00255125","term_id":"NCT00255125"}}NCT00255125     

Gene expression and biological pathways in tissue of men with prostate cancer in a randomized clinical trial of lycopene and fish oil supplementation

Background

Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial.

Methods

Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients.

Results

Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001).

Conclusions

We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00402285","term_id":"NCT00402285"}}NCT00402285     

The ubiquitin peptidase UCHL1 induces G0/G1 cell cycle arrest and apoptosis through stabilizing p53 and is frequently silenced in breast cancer

Background

Breast cancer (BrCa) is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1) is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear.

Methodology/Principal Findings

We found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90%) and 53 of 66 (80%) primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells.

Conclusions/Significance

UCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.     

A Somatic MAP3K3 Mutation Is Associated with Verrucous Venous Malformation

Verrucous venous malformation (VVM), also called “verrucous hemangioma,” is a non-hereditary, congenital, vascular anomaly comprised of aberrant clusters of malformed dermal venule-like channels underlying hyperkeratotic skin. We tested the hypothesis that VVM lesions arise as a consequence of a somatic mutation. We performed whole-exome sequencing (WES) on VVM tissue from six unrelated individuals and looked for somatic mutations affecting the same gene in specimens from multiple persons. We observed mosaicism for a missense mutation ({"type":"entrez-nucleotide","attrs":{"text":"NM_002401.3","term_id":"42794764","term_text":"NM_002401.3"}}NM_002401.3, c.1323C>G; {"type":"entrez-protein","attrs":{"text":"NP_002392","term_id":"42794765","term_text":"NP_002392"}}NP_002392, p.Iso441Met) in mitogen-activated protein kinase kinase kinase 3 (MAP3K3) in three of six individuals. We confirmed the presence of this mutation via droplet digital PCR (ddPCR) in the three subjects and found the mutation in three additional specimens from another four participants. Mutant allele frequencies ranged from 6% to 19% in affected tissue. We did not observe this mutant allele in unaffected tissue or in affected tissue from individuals with other types of vascular anomalies. Studies using global and conditional Map3k3 knockout mice have previously implicated MAP3K3 in vascular development. MAP3K3 dysfunction probably causes VVM in humans.     

Circulating Tumor Cells Count and Morphological Features in Breast,Colorectal and Prostate Cancer

Background

Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters.

Methodology

Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival.

Results

Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected.

Significance of the study

CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer.     

An integrated approach of bioinformatic prediction and in vitro analysis identified that miR-34a targets <Emphasis Type="Italic">MET</Emphasis> and <Emphasis Type="Italic">AXL</Emphasis> in triple-negative breast cancer

Background

Breast cancer is the most prevalent cancer among women, and AXL and MET are the key genes in the PI3K/AKT/mTOR pathway as critical elements in proliferation and invasion of cancer cells. MicroRNAs (miRNAs) are small non-coding RNAs regulating the expression of genes.

Methods

Bioinformatic approaches were used to find a miRNA that simultaneously targets both AXL and MET 3′-UTRs. The expression of target miRNA was evaluated in triple-negative (MDA-MB-231) and HER2-overexpressing (SK-BR-3) breast cancer cell lines as well as normal breast cells, MCF-10A, using quantitative real-time PCR. Then, the miRNA was overexpressed in normal and cancer cell lines using a lentiviral vector system. Afterwards, effects of overexpressed miRNA on the expression of AXL and MET genes were evaluated using quantitative real-time PCR.

Results

By applying bioinformatic software and programs, miRNAs that target the 3′-UTR of both AXL and MET mRNAs were determined, and according to the scores, miR-34a was selected for further analyses. The expression level of miR-34a in MDA-MB-231 and SK-BR-3 was lower than that of MCF-10A. Furthermore, AXL and MET expression in SK-BR-3 and MDA-MB-231 was lower and higher, respectively, than that of MCF-10A. After miR-34a overexpression, MET and AXL were downregulated in MDA-MB-231. In addition, MET was downregulated in SK-BR-3, while AXL was upregulated in this cell line.

Conclusions

These findings may indicate that miR-34a is an oncogenic miRNA, downregulated in the distinct breast cancer subtypes. It also targets MET and AXL 3′-UTRs in triple-negative breast cancer. Therefore, it can be considered as a therapeutic target in this type of breast cancer.

     

Sprouty 2 is an independent prognostic factor in breast cancer and may be useful in stratifying patients for trastuzumab therapy

Background

Resistance to trastuzumab is a clinical problem, partly due to overriding activation of MAPK/PI3K signalling. Sprouty-family proteins are negative regulators of MAPK/PI3K signalling, but their role in HER2-therapy resistance is unknown.

Patients and Methods

Associations between Sprouty gene expression and clinicopathological features were investigated in a breast cancer microarray meta-analysis. Changes in expression of Spry2 and feedback inhibition on trastuzumab resistance were studied in SKBr3 and BT474 breast carcinoma cell lines using cell viability assays. Spry2 protein expression was measured by quantitative immunofluorescence in a cohort of 122 patients treated with trastuzumab.

Results

Low gene expression of Spry2 was associated with increased pathological grade, high HER2 expression, and was a significant independent prognostic factor. Overexpression of Spry2 in SKBr3s resulted in enhanced inhibition of cell viability after trastuzumab treatment, and the PI3K-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 had a similar effect. Low Spry2 expression was associated with increased risk of death (HR = 2.28, 95% CI 1.22–4.26; p = 0.008) in trastuzumab-treated patients, including in multivariate analysis. Stratification of trastuzumab-treated patients using PTEN and Spry2 was superior to either marker in isolation.

Conclusion

In breast cancers with deficient feedback inhibition, combinatorial therapy with negative regulators of growth factor signalling may be an effective therapeutic strategy.     

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO₂ fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg²⁺ for catalysis and, like RuBisCO, was stimulated by CO₂. Four residues that are essential for the enolization reaction of RuBisCO, Lys¹⁷⁵, Lys²⁰¹, Asp²⁰³, and Glu²⁰⁴, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys¹²³, the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K_i value of 103 μm. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)⁴ catalyzes the carboxylation and oxygenation reactions of ribulose 1,5-bisphosphate (RuBP) in photosynthesis (1-4). This enzyme is the sole CO₂-fixing enzyme in plants; however, it has certain inefficiencies. It has a very low turnover rate, a low affinity for the substrate, CO₂, and low specificity between the carboxylation and oxygenation reactions (5-7). Thus, the intrinsic enzymatic properties of RuBisCO are inadequate for efficient incorporation of CO₂ into organic matter in photosynthesis (7). However, plants have overcome these disadvantages by investing a huge amount of leaf nitrogen in RuBisCO synthesis (8).In nature, there are wide variations in the properties and primary sequences of RuBisCO among different photosynthetic organisms (9-12). The primary sequences vary as much as 73% without loss of activity. The relative specificity ranges from ∼0.5 in a small subunitless RuBisCO to 238 in a red algal, hexadecameric RuBisCO (13, 14). The affinity for CO₂ varies some 100-fold (15). Comparisons between these kinetic parameters and the primary sequences are expected to reveal promising strategies for improving the enzyme, and many studies have been conducted on this topic (7, 16-18).A RuBisCO-like protein (RLP) with no CO₂-fixing activity was first demonstrated in Chlorobium tepidum (19), and a similar protein in Bacillus subtilis was found to be involved in the methionine salvage pathway (20). These findings have pointed to a new direction in RuBisCO research (17, 21). The phylogenetic tree of the catalytic subunits of RuBisCOs and their homologs shows four major clusters, forms I-III, and form IV (Fig. 1A). Form I and II RuBisCOs are involved in photosynthetic or chemosynthetic CO₂ fixation, whereas the metabolic function of form III RuBisCOs remains unclear, although they can fix CO₂ on RuBP (9, 22). Forms I-III conserve almost all 19 amino acid residues that are essential for CO₂ fixation in RuBisCO (Fig. 1B). The form IV cluster in the phylogenetic tree consists of RLPs that show ∼20% homology to plant form I or bacterial form II RuBisCOs (12, 20, 21, 23-25). There are 8-18 RuBisCO-essential residues that are conserved in RLPs (Fig. 1B). Form IV RLPs are further subdivided into four groups; α1, α2, β, and γ (21). The RLP of B. subtilis is classified in α1 and catalyzes the enolization reaction of 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) but not the carboxylation of RuBP (Fig. 2A) (20, 21, 23). The absence of CO₂-fixing activity in the B. subtilis RLP may be ascribed to changes in 8 of the 19 amino acid residues essential for CO₂ fixation in RuBisCO (Fig. 1B). Several of these residues are located at the C-terminal domains of B. subtilis RLP and RuBisCO. The dimeric RuBisCO from Rhodospirillum rubrum catalyzes the DK-MTP-1-P enolase reaction with very low activity (20). These findings, together with the similarity in the chemical structures of substrates for B. subtilis RLP and RuBisCO (Fig. 2A), suggest that they may have a close evolutionary relationship (12, 21, 23-25).Open in a separate windowFIGURE 1.Homology between RLPs and RuBisCOs. A, phylogenetic tree of RLPs and RuBisCOs. Deduced amino acid sequence of B. subtilis subsp. subtilis str. 168 RLP ({"type":"entrez-protein","attrs":{"text":"NP_389242","term_id":"16078423","term_text":"NP_389242"}}NP_389242) was compared with sequences of RLPs of Thermotoga lettingae TMO ({"type":"entrez-protein","attrs":{"text":"YP_001471302","term_id":"157364535","term_text":"YP_001471302"}}YP_001471302), Beggiatoa sp. SS ({"type":"entrez-protein","attrs":{"text":"ZP_01997270","term_id":"153864333","term_text":"ZP_01997270"}}ZP_01997270), Ostreococcus tauri (Ostreococcus tauri IV, {"type":"entrez-protein","attrs":{"text":"CAL54998","term_id":"116059291","term_text":"CAL54998"}}CAL54998), Alkalilimnicola ehrlichei MLHE-1 ({"type":"entrez-protein","attrs":{"text":"YP_742007","term_id":"114320324","term_text":"YP_742007"}}YP_742007), R. rubrum ATCC 11170 (R. rubrum IV, {"type":"entrez-protein","attrs":{"text":"YP_427085","term_id":"83593333","term_text":"YP_427085"}}YP_427085), R. palustris CGA009 (R. palustris IV-1, {"type":"entrez-protein","attrs":{"text":"NP_947514","term_id":"39935238","term_text":"NP_947514"}}NP_947514), Archaeoglobus fulgidus DSM 4304 (A. fulgidus IV, {"type":"entrez-protein","attrs":{"text":"NP_070416","term_id":"11499182","term_text":"NP_070416"}}NP_070416), M. aeruginosa PCC 7806 (M. aeruginosa IV, {"type":"entrez-protein","attrs":{"text":"CAJ43366","term_id":"112821115","term_text":"CAJ43366"}}CAJ43366), G. kaustophilus HTA426 ({"type":"entrez-protein","attrs":{"text":"YP_146806","term_id":"56419488","term_text":"YP_146806"}}YP_146806), Bacillus cereus ATCC 14579 ({"type":"entrez-protein","attrs":{"text":"NP_833754","term_id":"30022123","term_text":"NP_833754"}}NP_833754), B. bronchiseptica RB50 ({"type":"entrez-protein","attrs":{"text":"NP_887583","term_id":"33600023","term_text":"NP_887583"}}NP_887583), Polaromonas sp. JS666 ({"type":"entrez-protein","attrs":{"text":"YP_546958","term_id":"91786006","term_text":"YP_546958"}}YP_546958), C. tepidum TLS ({"type":"entrez-protein","attrs":{"text":"NP_662651","term_id":"21674586","term_text":"NP_662651"}}NP_662651), and R. palustris CGA009 (R. palustris IV-2, {"type":"entrez-protein","attrs":{"text":"NP_945615","term_id":"39933339","term_text":"NP_945615"}}NP_945615) and of RuBisCOs of R. palustris CGA009 (R. palustris II, {"type":"entrez-protein","attrs":{"text":"NP_949975","term_id":"39937699","term_text":"NP_949975"}}NP_949975), R. rubrum ATCC 11170 (R. rubrum II, {"type":"entrez-protein","attrs":{"text":"YP_427487","term_id":"83593735","term_text":"YP_427487"}}YP_427487), M. jannaschii DSM 2661 ({"type":"entrez-protein","attrs":{"text":"NP_248230","term_id":"15669420","term_text":"NP_248230"}}NP_248230), A. fulgidus DSM 4304 (A. fulgidus III, {"type":"entrez-protein","attrs":{"text":"NP_070466","term_id":"11499228","term_text":"NP_070466"}}NP_070466), Thermococcus kodakaraensis KOD1 ({"type":"entrez-protein","attrs":{"text":"YP_184703","term_id":"57642225","term_text":"YP_184703"}}YP_184703), Galdieria partita ({"type":"entrez-protein","attrs":{"text":"BAA75796","term_id":"4519903","term_text":"BAA75796"}}BAA75796), R. palustris CGA009 (R. palustris I, {"type":"entrez-protein","attrs":{"text":"NP_946905","term_id":"39934629","term_text":"NP_946905"}}NP_946905), M. aeruginosa PCC 7806 (M. aeruginosa I, {"type":"entrez-protein","attrs":{"text":"CAJ43363","term_id":"112821111","term_text":"CAJ43363"}}CAJ43363), O. tauri (O. tauri IV, {"type":"entrez-protein","attrs":{"text":"YP_717262","term_id":"113170470","term_text":"YP_717262"}}YP_717262), and S. oleracea ({"type":"entrez-protein","attrs":{"text":"NP_054944","term_id":"11497536","term_text":"NP_054944"}}NP_054944). When an organism has more than one RuBisCO and/or RLP sequence, the form number of each sequence in the RuBisCO family follows the name of the organism. ClustalW and TreeView programs (available on the World Wide Web) were used to construct the phylogenetic tree. B, multiple alignments of sequences underlined in A. Identical amino acid residues are indicated by black shading, and similar amino acid residues are indicated by gray shading. Sequences are numbered according to the S. oleracea sequence. Catalytic and RuBP-binding residues deduced for RuBisCO are indicated by open triangles and filled triangles, respectively. Alignment was visualized with the BOXSHADE program (available on the World Wide Web).Open in a separate windowFIGURE 2.Catalytic and structural similarity of RLPs and RuBisCOs. A, catalytic reactions of RuBisCO and RLP. B, comparison of active sites between S. oleracea RuBisCO binding CABP (8RUC) and G. kaustophilus RLP (2OEM) modeled to bind DK-MTP-1-P. DK-MTP-1-P in G. kaustophilus RLP was depicted by substituting the methyl group of DK-H-1-P in 2OEM with the thiomethyl group of MTRu-1-P bound to MtnA (28). Side chains of active site residues and ligands are shown as sticks. These five residues of B. subtilis were substituted with other amino acids in this study. CABP and DK-MTP-1-P are shown in white, and their phosphate groups are shown in red and orange, respectively. Mg²⁺ atoms are shown in yellow. Protein structures were drawn with PyMOL (available on the World Wide Web).The RuBisCO reaction starts with the abstraction of the C3 proton from RuBP to form the cis-enediol(ate) of RuBP (Fig. 2A) (26). Using the spinach numbering format to identify RuBisCO and RLP residues, the carbamate formed on the ε-amino group of Lys²⁰¹ may be the general base to abstract the proton, and the cis-enediol(ate) form of RuBP is stabilized in the combination of side chains from Lys¹⁷⁵ and His²⁹⁴ (27). Asp²⁰³, Glu²⁰⁴, and the carbamate Lys²⁰¹ of the enzyme active site stabilize the cis-enediol(ate) and CO₂ through the Mg²⁺ ion (26). The B. subtilis RLP abstracts the C1 proton of its substrate DK-MTP-1-P to start the DK-MTP-1-P enolization reaction (12, 21, 23). The ε-amino group of Lys¹²³ is thought to be required for the abstraction of the 1-proS proton in the Geobacillus kaustophilus RLP, which belongs to group α1, together with the B. subtilis RLP (Fig. 2B) (25). Lys¹²³ is conserved among DK-MTP-1-P enolases and resides very near the C1 of 2,3-diketohexane 1-phosphate (DK-H-1-P), a structural analogue of DK-MTP-1-P. As is the case in RuBisCO, the enolate intermediate is stabilized by Mg²⁺ and several amino acid residues: Lys¹⁷⁵, Asp²⁰³, Glu²⁰⁴, His²⁹⁴, and the carbamylated Lys²⁰¹.The results of these studies suggest that the DK-MTP-1-P enolase is structurally and functionally related to photosynthetic RuBisCO. However, research on the G. kaustophilus RLP revealed that the proton-abstracting, reaction-starting residues differed between the DK-MTP-1-P enolase and RuBisCO (25). It has been reported that when lysine at 201 is substituted with an alanine in the G. kaustophilus RLP, the enzyme is still capable of catalyzing enolization of DK-MTP-1-P (25). This result raises a question about the above hypothesis on the close evolutionary relationship between the RLP and RuBisCO, because a carbamylated lysine residue would be required at this position to form the Mg²⁺-chelating triad linkage together with Asp²⁰³ and Glu²⁰⁴ and to stabilize the reaction intermediate in the RuBP enolization reaction of RuBisCO.Evolutionary relationships of genes with similar sequences are deduced by comparing gene sequence homology of the genes and amino acid sequence homology of the predicted proteins and by analyzing conservation of functional motifs of the predicted proteins in silico. Comparison of protein structures at the active sites also provides important information. However, it may difficult to predict their mutual evolutionary relationship more precisely when they catalyze different reactions in individual metabolic pathways. The present research adopted a new method to resolve such an issue.We studied the structural and functional interrelationships of RLP and RuBisCO after enzymological characterization of B. subtilis RLP as the DK-MTP-1-P enolase enzyme. The results showed that DK-MTP-1-P enolase activity was limited to some RLPs in the cluster, including B. subtilis in form IV RLPs. All of the catalytic residues for the RuBisCO reaction were also indispensable for DK-MTP-1-P enolase activity. The architecture of the B. subtilis RLP substrate-binding residues stereospecifically stabilized the transition state analog in CO₂ fixation of RuBisCO. The fact that the transition state analog of RuBisCO interacts with the active site of Bacillus RLP strongly supports their evolutionary proximity.