Structural and Functional Characterization of Mycobacterium tuberculosis Homoserine Transacetylase.

Mycobacterium tuberculosis (Mtb) lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essential S-adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validated Mtb HTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization of Mtb HTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substrates─acetyl-CoA and l-homoserine─engage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA and O-acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractive Mtb target.

Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208-215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the "new Pi site" attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.

Infrared thermal imaging-based research on the intermediate structures of the lung and large intestine exterior-interior relationship in asthma patients.

OBJECTIVE: By observing body surface temperature variation of the intermediate structures of the Lung (Fei) and Large Intestine (Dachang) exterior-interior relationship in asthmatic patients, to investigate the pathological response on the pathway of channels and to substantiate the objective existence of the intermediary structures. METHODS: The study included 60 subjects meeting the bronchial asthma inclusion criteria (experimental group) and 60 healthy subjects (normal control group). ATIR-M301 infrared thermal imaging device was used for detecting body surface temperature of the subjects and collecting the infrared thermal images. The temperature values of the intermediate structures of Lung and Large Intestine exterior-interior relationship [throat, Quepen, elbow, nose, Lieque (LU 7), Pianli (LI 6)], control areas (0.2 cm lateral to the above structures) and Yintang (EX-HN 3) were measured on the infrared thermal image by infrared imaging system. Then, the above temperature values were compared and analyzed within and between two groups. RESULTS: There were insignificant differences between the temperature on the left and right sides of the intermediate structures (Quepen, elbow, LU 7, LI 6) in normal control group (P>0.05). Except for that of Quepen, there were insignifificant differences between the temperature of the intermediate structures and their corresponding control areas in normal control group (P>0.05). In the experimental group, the temperature on the left and right sides of the intermediate structures (Quepen, elbow, LU 7, LI 6) showed statistically signifificant differences (P<0.05 or P<0.01); the temperature difference between intermediate structure (throat, Quepen, elbow, nose, LI 6) and their respective control areas were also significant (P<0.05 or P<0.01). The temperature of the intermediate structures (throat, Quepen, elbow, LU7, LI 6) between the experimental group and normal control group showed signifificant differences (P<0.05 or P<0.01). CONCLUSIONS: This study is an initial step to validate the objective existence of Lung and Large Intestine exterior-interior relationship intermediate structures, as described in the Chinese classical medical literatures, through the functional imaging angle. The intermediate structures are the pathological reaction areas of the bronchial asthmatic patients.

Infrared thermal imaging-based research on the intermediate structures of the lung and large intestine exterior-interior relationship in asthma patients / 中国结合医学杂志

<p><b>OBJECTIVE</b>By observing body surface temperature variation of the intermediate structures of the Lung (Fei) and Large Intestine (Dachang) exterior-interior relationship in asthmatic patients, to investigate the pathological response on the pathway of channels and to substantiate the objective existence of the intermediary structures.</p><p><b>METHODS</b>The study included 60 subjects meeting the bronchial asthma inclusion criteria (experimental group) and 60 healthy subjects (normal control group). ATIR-M301 infrared thermal imaging device was used for detecting body surface temperature of the subjects and collecting the infrared thermal images. The temperature values of the intermediate structures of Lung and Large Intestine exterior-interior relationship [throat, Quepen, elbow, nose, Lieque (LU 7), Pianli (LI 6)], control areas (0.2 cm lateral to the above structures) and Yintang (EX-HN 3) were measured on the infrared thermal image by infrared imaging system. Then, the above temperature values were compared and analyzed within and between two groups.</p><p><b>RESULTS</b>There were insignificant differences between the temperature on the left and right sides of the intermediate structures (Quepen, elbow, LU 7, LI 6) in normal control group (P>0.05). Except for that of Quepen, there were insignifificant differences between the temperature of the intermediate structures and their corresponding control areas in normal control group (P>0.05). In the experimental group, the temperature on the left and right sides of the intermediate structures (Quepen, elbow, LU 7, LI 6) showed statistically signifificant differences (P<0.05 or P<0.01); the temperature difference between intermediate structure (throat, Quepen, elbow, nose, LI 6) and their respective control areas were also significant (P<0.05 or P<0.01). The temperature of the intermediate structures (throat, Quepen, elbow, LU7, LI 6) between the experimental group and normal control group showed signifificant differences (P<0.05 or P<0.01).</p><p><b>CONCLUSIONS</b>This study is an initial step to validate the objective existence of Lung and Large Intestine exterior-interior relationship intermediate structures, as described in the Chinese classical medical literatures, through the functional imaging angle. The intermediate structures are the pathological reaction areas of the bronchial asthmatic patients.</p>