scholarly journals Role of Homoserine Transacetylase as a New Target for Antifungal Agents

2007 ◽  
Vol 51 (5) ◽  
pp. 1731-1736 ◽  
Author(s):  
Ishac Nazi ◽  
Adam Scott ◽  
Anita Sham ◽  
Laura Rossi ◽  
Peter R. Williamson ◽  
...  
Keyword(s):  
Small Molecules ◽  
Antifungal Agents ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Small Molecule Inhibition ◽  
Steady State Kinetic ◽  
Antibiotic Discovery ◽  
Homoserine Transacetylase ◽  
State Kinetic

ABSTRACT Microbial amino acid biosynthesis is a proven yet underexploited target of antibiotics. The biosynthesis of methionine in particular has been shown to be susceptible to small-molecule inhibition in fungi. The first committed step in Met biosynthesis is the acylation of homoserine (Hse) by the enzyme homoserine transacetylase (HTA). We have identified the MET2 gene of Cryptococcus neoformans H99 that encodes HTA (CnHTA) by complementation of an Escherichia coli metA mutant that lacks the gene encoding homoserine transsuccinylase (HTS). We cloned, expressed, and purified CnHTA and determined its steady-state kinetic parameters for the acetylation of L-Hse by acetyl coenzyme A. We next constructed a MET2 mutant in C. neoformans H99 and tested its growth behavior in Met-deficient media, confirming the expected Met auxotrophy. Furthermore, we used this mutant in a mouse inhalation model of infection and determined that MET2 is required for virulence. This makes fungal HTA a viable target for new antibiotic discovery. We screened a 1,000-compound library of small molecules for HTA inhibitors and report the identification of the first inhibitor of fungal HTA. This work validates HTA as an attractive drug-susceptible target for new antifungal agent design.

scholarly journals New standards for collecting and fitting steady state kinetic data

2019 ◽  
Vol 15 ◽  
pp. 16-29 ◽  
Author(s):  
Kenneth A Johnson
Keyword(s):  
Conformational Changes ◽  
Short Review ◽  
Order Rate Constant ◽  
Rate Equations ◽  
Enzyme Specificity ◽  
Additional Information ◽  
Steady State Kinetic ◽  
Specificity Constant ◽  
State Kinetic

The Michaelis–Menten equation is usually expressed in terms of k cat and K m values: v = k cat[S]/(K m + [S]). However, it is impossible to interpret K m in the absence of additional information, while the ratio of k cat/K m provides a measure of enzyme specificity and is proportional to enzyme efficiency and proficiency. Moreover, k cat/K m provides a lower limit on the second order rate constant for substrate binding. For these reasons it is better to redefine the Michaelis–Menten equation in terms of k cat and k cat/K m values: v = k SP[S]/(1 + k SP[S]/k cat), where the specificity constant, k SP = k cat/K m. In this short review, the rationale for this assertion is explained and it is shown that more accurate measurements of k cat/K m can be derived directly using the modified form of the Michaelis–Menten equation rather than calculated from the ratio of k cat and K m values measured separately. Even greater accuracy is achieved with fitting the raw data directly by numerical integration of the rate equations rather than using analytically derived equations. The importance of fitting to derive k cat and k cat/K m is illustrated by considering the role of conformational changes in enzyme specificity where k cat and k cat/K m can reflect different steps in the pathway. This highlights the pitfalls in attempting to interpret K m, which is best understood as the ratio of k cat divided by k cat/K m.

scholarly journals Unravelling ceftazidime/avibactam resistance of KPC-28, a KPC-2 variant lacking carbapenemase activity

2019 ◽  
Vol 74 (8) ◽  
pp. 2239-2246 ◽  
Author(s):  
Saoussen Oueslati ◽  
Bogdan I Iorga ◽  
Linda Tlili ◽  
Cynthia Exilie ◽  
Agustin Zavala ◽  
...  
Keyword(s):  
Amino Acid ◽  
Susceptibility Testing ◽  
Fold Increase ◽  
Hydrolytic Activity ◽  
E Coli ◽  
Steady State Kinetic ◽  
Double Amino Acid ◽  
Or Gene ◽  
State Kinetic

Abstract Background KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions. Objectives To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243). Methods The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity. Results Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate. Conclusions We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection.

Recognition of aminoglycoside antibiotics by enterococcal-staphylococcal aminoglycoside 3'-phosphotransferase type IIIa: role of substrate amino groups.

1996 ◽  
Vol 40 (11) ◽  
pp. 2648-2650 ◽  
Author(s):  
G A McKay ◽  
J Roestamadji ◽  
S Mobashery ◽  
G D Wright
Keyword(s):  
Steady State ◽  
Aminoglycoside Antibiotics ◽  
Hydroxyl Group ◽  
Amino Group ◽  
Amino Groups ◽  
Type Iiia ◽  
Steady State Kinetic ◽  
State Kinetic

The interactions of the aminoglycoside 3'-phosphotransferase IIIa with aminoglycoside antibiotics lacking specific amino groups were examined by steady-state kinetic analyses. The results demonstrate that an amino group on C-1 and either an amino or a hydroxyl group at the 2' and 6' positions are important for detoxification of aminoglycosides by this enzyme.

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