Hydroxyl Groups in the ββ Sandwich of Metallo-β-lactamases Favor Enzyme Activity: A Computational Protein Design Study

Oelschlaeger, Peter; Mayo, Stephen L.

doi:10.1016/j.jmb.2005.04.044

Cited by 31 publications

(52 citation statements)

References 28 publications

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“…For the IMP-1-F218Y mutant, increased activity was observed (28,29), and this was also found for our tVIM-7-F218Y experiments (Table 3). The tVIM-7-F218Y mutant had better penicillin and ampicillin binding properties in terms of lower K m values, and the catalytic efficiencies against the tested cephalosporins were 8 (cefepime), 12 (ceftazidime), 9 (cefoxitin), and 10 (cefuroxime) times higher than those for the tVIM-7 wild type.…”

Section: R-sym ϭ {[⌺ H ⌺ I | I I (H) ϫ ͗I(h)͘|]/[⌺ H ⌺ I I(h)]} Whersupporting

confidence: 88%

“…To study the involvement of these residues and the SAR in VIM-7, we mutated these residues to tyrosine residues as they appear in VIM-2. For residue 218, the assumptions were based on studies of the IMP-1-F218Y mutant, which showed increased catalytic efficiency compared to the wild type through improved movements of the lid that covers the active site (28,29). The lid includes the two anti-parallel ␤-strands connected by the L1 loop.…”

Section: R-sym ϭ {[⌺ H ⌺ I | I I (H) ϫ ͗I(h)͘|]/[⌺ H ⌺ I I(h)]} Whermentioning

confidence: 99%

“…Two anti-parallel ␤-strands connected by a loop determine the L1 loop (residues 60 to 66) and are proposed to form a lid over the active site in several class B1 MBLs, including GIM-1 (27), VIM-7 (16), and IMP-1 (28)(29)(30). For the latter, the mutation of F218Y increased the catalytic efficiency compared to that of wild-type IMP-1 through hydrogen bond formation, improving the closingdown movement of this lid (28,29).…”

mentioning

confidence: 99%

“…For the latter, the mutation of F218Y increased the catalytic efficiency compared to that of wild-type IMP-1 through hydrogen bond formation, improving the closingdown movement of this lid (28,29). We therefore decided to make the same mutation in VIM-7 (F218Y) to analyze the effect of additional hydrogen bonds on the structure and enzyme activity despite the low amino acid sequence identity between VIM-7 and IMP-1 of 28%.…”

mentioning

confidence: 99%

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His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Leiros

Skagseth

Edvardsen

et al. 2014

Antimicrob Agents Chemother

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b Metallo-␤-lactamases (MBLs) are the causative mechanism for resistance to ␤-lactams, including carbapenems, in many Gramnegative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T m ) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher T m (57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T m , 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

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Section: R-sym ϭ {[⌺ H ⌺ I | I I (H) ϫ ͗I(h)͘|]/[⌺ H ⌺ I I(h)]} Whersupporting

confidence: 88%

Section: R-sym ϭ {[⌺ H ⌺ I | I I (H) ϫ ͗I(h)͘|]/[⌺ H ⌺ I I(h)]} Whermentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Leiros

Skagseth

Edvardsen

et al. 2014

Antimicrob Agents Chemother

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“…For the redesign of E. coli chorismate mutase, one of five single mutations predicted to maintain activity increased efficiency by 60% [42]. Separately, redesign of the imipenemase IMP-1 predicted a mutation that removes a hydroxyl group, and a double mutation that transfers the hydroxyl group [45]. Hydroxyl transfer altered substrate specificity, whereas the presence of both hydroxyl groups turned out to increase catalytic efficiency.…”

Section: Enzymesmentioning

confidence: 99%

Progress in computational protein design

Lippow

Tidor

2007

Current Opinion in Biotechnology

191

165

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Current progress in computational structure-based protein design is reviewed in the areas of methodology and applications. Foundational advances include new potential functions, more efficient ways of computing energetics, flexible treatments of solvent, and useful energy function approximations, as well as ensemble-based approaches to scoring designs for inclusion of entropic effects, improvements to guaranteed and to stochastic search techniques, and development of methods to design combinatorial libraries for screening and selection. Applications include new approaches and successes in the design of specificity for protein folding, binding, and catalysis, in the redesign of proteins for enhanced binding affinity, and in the application of design technology to study and alter enzyme catalysis. Computational protein design continues to mature and its future is bright.

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Minimale Umgestaltung aktiver Enzymtaschen – wie man alten Enzymen neue Kunststücke beibringt

2007

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Während die Natur ihre Katalysatoren über Millionen von Jahren entwickelt hat, versuchen Enzymingenieure, dies ein wenig schneller zu bewerkstelligen. Die aktive Tasche eines Enzyms bildet eine stark optimierte Mikroumgebung für die Katalyse chemischer Transformationen in biologischen Systemen – folglich können Änderungen in diesen Zentren die Enzymaktivität stark beeinflussen. Daher bietet die Voraussage und Kontrolle solcher Effekte einen vielversprechenden Weg, um zu neuen Funktionen zu gelangen. Der Ansatz, bei dem minimale Modifikationen in den aktiven Taschen von Enzymen (z. B. durch gezielte Mutagenese und Hinzufügen neuer reaktiver Funktionalitäten) deren katalytisches Repertoire erweitern sollen, ist der Schwerpunkt dieses Aufsatzes. Dabei kann eine neue Aktivität oft schon durch eine einzige Mutation erreicht werden. Die vielen erfolgreichen Beispiele für die Umgestaltung von aktiven Taschen durch minimale Mutationen geben wertvolle Einblicke in die Evolution von Enzymen und bahnen neue Wege in der Erforschung der Biokatalysatoren.

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Hydroxyl Groups in the ββ Sandwich of Metallo-β-lactamases Favor Enzyme Activity: A Computational Protein Design Study

Cited by 31 publications

References 28 publications

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Progress in computational protein design

Minimale Umgestaltung aktiver Enzymtaschen – wie man alten Enzymen neue Kunststücke beibringt

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