Amino Acid Sequence Determinants of β-Lactamase Structure and Activity

Huang, Wanzhi; Petrosino, Joseph F.; Hirsch, Marc; Shenkin, Peter S.; Palzkill, Timothy

doi:10.1006/jmbi.1996.0279

Cited by 166 publications

(207 citation statements)

References 0 publications

Supporting

Mentioning

197

Contrasting

Order By: Relevance

“…A deliberate comparison between natural and experimental variation was engineered in b-lactamase (Huang et al 1996;Palzkill & Botstein 1992). Site-directed mutagenesis was used to randomize codons systematically, two at a time, across the entire molecule.…”

Section: (C) Common Selectionmentioning

confidence: 99%

Experimental evolution recapitulates natural evolution

Wichman

Scott

Yarber

et al. 2000

Phil. Trans. R. Soc. Lond. B

View full text Add to dashboard Cite

Genomes of the closely related bacteriophages fX174 and S13 are 5386 bases long and di¡er at 114 nucleotides, a¡ecting 28 amino acids. Both parental phages were adapted to laboratory culture conditions in replicate lineages and analysed for nucleotide changes that accumulated experimentally. Of the 126 experimental substitutions, 90% encoded amino-acid changes, and 62% of the substitutions occurred in parallel in more than one exp erimental line. Furthermore, missense changes at 12 of the experimental sites were at residues di¡ering between the parental phages; in ten cases the fX174 experimental lineages were convergent with the S13 parent, or vice versa, at both the nucleotide and amino-acid levels. Convergence at a site was even obtained in both directions in three cases. These results point to a limited number of pathways taken during evolution in these viruses, and also raise the possibility that much of the amino-acid variation in the natural evolution of these viruses has been selected.

show abstract

Section: (C) Common Selectionmentioning

confidence: 99%

Experimental evolution recapitulates natural evolution

Wichman

Scott

Yarber

et al. 2000

Phil. Trans. R. Soc. Lond. B

View full text Add to dashboard Cite

show abstract

“…3). 24,34 A k* value of 1.0 indicates only one amino acid is observed after selection for function and signifies a position with stringent sequence requirements. Higher k* values indicate progressively more amino acids can be tolerated with preservation of function and represent positions with relaxed sequence requirements.…”

Section: Quantitation Of Random Substitution Results In Odcasementioning

confidence: 99%

“…Higher k* values indicate progressively more amino acids can be tolerated with preservation of function and represent positions with relaxed sequence requirements. 24,34 When the random libraries were selected for function in the context of the multicopy pTP304 plasmid, functional clones were found with substitutions at the Lys44-Asp71-Lys73-Asp76 positions with k* values ranging from 1.4 to 3.6 (Table II). In contrast, when the libraries were selected from the chromosomal location, only the wild-type amino acids were found for these positions (k* ¼ 1.0; Table II).…”

Section: Quantitation Of Random Substitution Results In Odcasementioning

confidence: 99%

“…The sequence requirements at a position in an enzyme can be determined experimentally by using a codon randomization and selection strategy. 24,25 For this approach, individual codons in the gene of interest are randomized using site-directed mutagenesis to create libraries containing all possible substitutions at a position(s). The sequence requirements at each position for 24 residue positions in the active site of ODCase was assessed by randomizing the amino acid sequence at each position and selecting for enzyme function by using an in vivo genetic selection.…”

Section: Introductionmentioning

confidence: 99%

“…The sequence requirements at each position for 24 residue positions in the active site of ODCase was assessed by randomizing the amino acid sequence at each position and selecting for enzyme function by using an in vivo genetic selection. [24][25][26] Interestingly, this very proficient active site is tolerant to substitution with only modest drops in catalytic proficiency associated with active site substitutions.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Determination of the amino acid sequence requirements for catalysis by the highly proficient orotidine monophosphate decarboxylase

Yuan¹,

Cárdenas²,

Gilbert³

et al. 2011

Protein Science

Self Cite

View full text Add to dashboard Cite

Orotidine 5 0 -monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5 0 -monophosphate to uridine 5 0 -monophosphate during pyrimidine nucleotide biosynthesis. This enzyme is one of the most proficient known, exhibiting a rate enhancement of over 17 orders of magnitude over the uncatalyzed rate. An interesting question is whether the high proficiency of ODCase is associated with a highly optimized sequence of active site residues. This question was addressed by randomizing 24 residue positions in and around the active site of the E. coli ODCase (pyrF) by site-directed mutagenesis. The libraries of mutants were selected for function from a multicopy plasmid or by single-copy replacement at the pyrF locus on the E. coli chromosome. Stringent sequence requirements for function were found for the mutants expressed from the chromosomal pyrF locus. Six positions were not tolerant of substitutions and several others accepted very limited substitutions. In contrast, all positions could be substituted to some extent when the library mutants were expressed from a multicopy plasmid. For the conserved quartet of charged residues Lys44-Asp71-Lys73-Asp76, a cysteine substitution was found to provide function at positions 71 and 76. A lower pK a for both cysteine mutants supports a mechanism whereby the thiolate group of cysteine substitutes for the negatively charged aspartate side chain. The partial function mutants such as D71C and D76C exhibit reduced catalytic efficiency relative to wild type but nevertheless provide a rate enhancement of 15 orders of magnitude over the uncatalyzed rate indicating the catalytic proficiency of the enzyme is robust and tolerant of mutation.

show abstract