Structure of human β-glucuronidase reveals candidate lysosomal targeting and active-site motifs

Jain, Sanjeev; Drendel, William B.; Chen, Z.W; Mathews, F.S.; Sly, William S.; Grubb, Jeffrey H.

doi:10.1038/nsb0496-375

Cited by 215 publications

(189 citation statements)

References 47 publications

Supporting

Mentioning

178

Contrasting

Unclassified

Order By: Relevance

“…This region (nucleotides 1320 -1812) contains the three randomized codons and about half of the catalytic domain (12). The 14 selected clones collectively contained 10 of 20 amino acids at position 557, 6 amino acids at position 566, and 9 amino acids at position 568 (Table II).…”

Section: Resultsmentioning

confidence: 99%

“…As noted above, the human GUS protein has been crystallized; the amino acid sequences of the E. coli and human GUS homologs are 50% identical, with highly conserved active sites (12). The E. coli ␤-glucuronidase gene (gusA, formerly uidA) is more amenable to directed evolution, because it can be overexpressed at high levels in E. coli, enabling the detection of weak catalytic activities in reactions with ␤-xylosides and other non-native substrates.…”

mentioning

confidence: 99%

“…Their amino acid sequences are too divergent to align, but six conserved active site residues are superimposable ( Fig. 1) (11,12). Our goal is to understand how enzymes like GUS and XYL evolved to become so specific.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Geddie

Matsumura

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli ␤-glucuronidase (GUS) variants with increased ␤-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-␤-D-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a ϳ200-fold improvement over the wild type in reactivity (k cat /K m ) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Geddie

Matsumura

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Numbers in parentheses give the number of amino acid residues between the residue aligned to Asn-158 or Asn-350 and the actual asparagine of the nearest potential N-glycosylation site in amino acid residues. ␤-glucuronidase a ␤-hairpin loop may be the common structural determinant of the phosphotransferase recognition domain (12,13). The existence of a critical lysine residue in a helix in ASA demonstrates that this cannot be the case for all lysosomal enzymes.…”

Section: In Vitro Mutagenesis Of Lysine Residues-humanmentioning

confidence: 99%

“…Comparisons of three-dimensional structures of non-homologous cathepsins A, B, and D in combination with peptide inhibition experiments of in vitro phosphorylation indicated that a ␤-hairpin loop structure containing the relevant lysines may be an important common recognition determinant in the cathepsins and in ␤-glucuronidase (12,13). Comparable hairpin loops, however, where not found among those residues relevant for proper sorting of aspartylglucosaminidase (11).…”

mentioning

confidence: 99%

Recognition of Arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal Enzyme N-Acetylglucosamine-phosphotransferase

Yaghootfam

Schestag

Dierks

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.

show abstract

Structural trees for protein superfamilies

Ефимов

1997

Proteins

View full text Add to dashboard Cite

Structural trees for large protein superfamilies, such as beta proteins with the aligned beta sheet packing, beta proteins with the orthogonal packing of alpha helices, two-layer and three-layer alpha/beta proteins, have been constructed. The structural motifs having unique overall folds and a unique handedness are taken as root structures of the trees. The larger protein structures of each superfamily are obtained by a stepwise addition of alpha helices and/or beta strands to the corresponding root motif, taking into account a restricted set of rules inferred from known principles of the protein structure. Among these rules, prohibition of crossing connections, attention to handedness and compactness, and a requirement for alpha helices to be packed in alpha-helical layers and beta strands in beta layers are the most important. Proteins and domains whose structures can be obtained by stepwise addition of alpha helices and/or beta strands to the same root motif can be grouped into one structural class or a superfamily. Proteins and domains found within branches of a structural tree can be grouped into subclasses or subfamilies. Levels of structural similarity between different proteins can easily be observed by visual inspection. Within one branch, protein structures having a higher position in the tree include the structures located lower. Proteins and domains of different branches have the structure located in the branching point as the common fold.

show abstract

Structure of human β-glucuronidase reveals candidate lysosomal targeting and active-site motifs

Cited by 215 publications

References 47 publications

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Recognition of Arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal Enzyme N-Acetylglucosamine-phosphotransferase

Structural trees for protein superfamilies

Contact Info

Product

Resources

About