Reaction mechanism of N-cyclopropylglycine oxidation by monomeric sarcosine oxidase.

Monomeric sarcosine oxidase (MSOX) is a fundamental - yet one of the simplest - member of a family of flavoenzymes able to catalyze the oxidation of sarcosine (N-methylglycine) and other secondary amines. MSOX is one of the best characterized members of the amine oxidoreductases (AOs), however, its reaction mechanism is still controversial. A single electron transfer (SET) process was suggested on the basis of studies with N-cyclopropylglycine (CPG), although a hydride transfer mechanism would be more consistent in general for AOs. To shed some light on the detailed reaction mechanisms of CPG in MSOX, we performed hybrid quantum mechanical/molecular mechanical (QM/MM) simulations. We found that the polar mechanism is energetically the most favorable. The free energy profile indicates that the first rate-limiting step is the CPG binding to the flavin ring which simultaneously proceeds with the ring-opening of the CPG cyclopropyl group. This reaction step of the CPG adduct formation corresponds to the nucleophilic attack of the cyclopropyl group (C3 atom) to the flavin ring (C4a atom), whereas the expected radical species formation in the SET mechanism was not observed. The following inactivated species, which accumulates during the CPG oxidation in MSOX, can be ascribed to an imine state, and not an enamine state, on the basis of the computed UV/Vis spectra. The conformation of CPG was found to be crucial for reactions following the CPG adduct formation.

The reaction mechanism of sarcosine oxidase elucidated using FMO and QM/MM methods.

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that oxidizes sarcosine to the corresponding imine product and is widely used in clinical diagnostics to test renal function. In the past decade, several experimental studies have been performed to elucidate the underlying mechanism of this oxidation reaction. However, the details of the molecular mechanism remain unknown. In this study, we theoretically examined three possible reaction mechanisms, namely, the single-electron transfer, hydride-transfer, and polar mechanisms, using the fragment molecular orbital (FMO) and mixed quantum mechanics/molecular mechanics (QM/MM) methods. We found that, of the three possible reaction pathways, hydride-transfer is the most energetically favorable mechanism. Significantly, hydrogen is not transferred in the hydride state (H-) but in a hydrogen atom state (H˙). Furthermore, a single electron is simultaneously transferred from sarcosine to flavin through their overlapping orbitals. Therefore, based on a detailed theoretical analysis of the calculated reaction pathway, the reaction mechanism of MSOX can be labeled the "hydrogen-atom-coupled electron-transfer" (HACET) mechanism instead of being categorized as the classical hydride-transfer mechanism. QM/MM and FMO calculations revealed that sarcosine is moved close to the flavin ring because of a small charge transfer (about 0.2 electrons in state 1 (MSOX-sarcosine complex)) and that the positively charged residues (Arg49, Arg52, and Lys348) near the active site play a prominent role in stabilizing the sarcosine-flavin complex. These results indicate that strong Coulombic interactions primarily control amine oxidation in the case of MSOX. The new reaction mechanism, HACET, will be important for all the flavoprotein-catalyzed oxidation reactions.

Microsatellite instability in poorly differentiated colorectal adenocarcinoma, particularly in relation to two subtypes.

BACKGROUND/AIMS: There have been a few reports indicating the characteristics of poorly differentiated colorectal adenocarcinoma by dividing it into subtypes. However, we have not found any reports describing microsatellite instability (MSI) of each subtype. In this study, we elucidated the clinicopathological features of subtypes in the poorly differentiated adenocarcinoma, especially the relationship between MSI and each subtype. METHODOLOGY: The present study included 28 cases with poorly differentiated adenocarcinoma. The 28 cases were classified into two groups; the solid group and the non-solid group. For each group, the clinicopathological aspects and MSI were examined. RESULTS: No significant differences were noted between the solid group and the non-solid group in terms of clinicopathological findings excluding male/female ratio. The 5-year survival rate of the solid group (38.5%) was significantly higher than that of the non-solid group (0.00%) (p=0.0013). The ratio of cases with MSI-H in the solid group (80.0%=12/15) was significantly higher than that of the non-solid group (30.8%=4/13) (p=0.0087). CONCLUSIONS: The incidence of MSI as well as the prognosis was different between solid and non-solid type with poorly differentiated colorectal adenocarcinoma. Therefore, we think that poorly differentiated colorectal adenocarcinoma should be classified into two subtypes: solid type and non-solid type when analysis for poorly differentiated adenocarcinoma is performed.

Two subtypes in colorectal mucinous carcinoma in relation to microsatellite instability.

BACKGROUND/AIMS: Although some investigators have attempted to divide colorectal mucinous adenocarcinoma into two entities, there have been few reports describing microsatellite instability of each subtype. In this study, we elucidated the clinicopathological features of subtypes in mucinous carcinoma, especially the relationship between microsatellite instability and each subtype. METHODOLOGY: The present study included 33 patients with mucinous colorectal cancer. The 33 patients were classified into two groups; the papillotubular type group (n = 22) and the mucocellular type group (n = 11). The clinicopathological aspects and microsatellite instability were examined. RESULTS: Significant differences were found between the papillotubular type group and the mucocellular type group regarding the following findings such as lymphatic invasion, lymph node metastasis, peritoneal metastasis, and Dukes stage. The mucocellular type group had 2 cases with high-frequency microsatellite instability, 7 cases with low-frequency microsatellite instability and 2 cases with microsatellite stability. Conversely, the papillotubular type group included 12 cases with high-frequency microsatellite instability, 3 cases with low-frequency microsatellite instability and 7 cases with microsatellite stability. The ratio of cases with high-frequency microsatellite instability in the mucocellular type group (18.1%) was significantly lower than that of the papillotubular type group (54.5%) (P = 0.0463). The 5-year survival rate of the mucocellular type group (29.1%) was significantly lower than that of the papillotubular type group (70.3%) (P = 0.0282). CONCLUSIONS: Colorectal mucinous carcinoma needs to be classified into two groups, papillotubular type and mucocellular type, because of significant differences in microsatellite instability and patients' survival.