Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase.

N Amuro,J L Paluh,H Zalkin

doi:10.1016/s0021-9258(17)38649-0

Abstract

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.

Highlights

In thisreport we present the initial resultsof an alternative glutaminyl-Cys-84 intermediate.Thedatafavorthe approach to identify residues that are essential for glutamine interpretation thaHt is-170 functionsas a general base amide transfer function
Reversion analysis is consistent with a function of His-170 in catalysis as Anthranilate synthaseis a glutamine amidotransferase that opposed to a structural role
For an amino acid that exerts a structural role, a different amino acid could be functional at theinitial siteor at a second site [27,28].E. coli strain JMBS(AtrpEGD) bearing plasmids trpE+GlD+(AS I1 Gly-84) or trpE+G2D+ (AS I1 Tyr-170) does not grow on minimal agar media containing 1mM NH&l because mutant anthranilate synthasewith defective glutamine amide transfer function cannot utilize glutamine for tryptophan synthesis

Summary

EXPERIMENTAL PROCEDURES

Of dissimilar subunits designated AS 1’ and AS I1 [1].AS I Strains, Plasmids, and Phage-Escherichia coli strain JMB9, rele-. Enzyme Purification-Wild type S. marcescens anthranilate synthase was purified from E. coli strainJMBS (AtrpEGD)/'pJP20 (trpE'G+D+) [7]. Anthranilate synthase having AS 11 Gly-84or Tyr170 replacements was purified from the same bacterial strain bearing plasmids pJPZl (trpE+GZD+)or pNA4 (trpE+GZD'), respectively. Selectionof Reuertunts-Strain JMBS (AtrpEGD)bearing plasmids pJP19 (trpE+GZD+)or pNA3 (trpE+GZD+)cannot synthesize tryptophan andgrow normally in M9 media [22],with 50 pg/ml ampicillin, containing 1 mM NH&1 as a nitrogen source because of defective anthranilate synthase glutamine amide transfer. The trpG2 mutation was transferred in vitro to plasmid pJP17 (trpE+G+D+)to yield pNA3 (trpE+G2D+)in which trp genes are transcribedfrom a pBR322 plasmid promoter. Transformants were screened on lowNH3 plates containing ampicillin, and representative clones were checked by enzyme assay, measurement of growth rate, and DNA sequencing. To confirm that the Tyr-170 replacement inactimixtures as determined by DNA sequencing

RESULTS

G Wild Type t r p G 2

DISCUSSION

HYDROLYSIS OF THIOESTER