165:
229:
200:
The catalytic mechanism of FGE is well studied. A multistep redox reaction with a covalent enzyme:substrate intermediate is proposed. The role of the cysteine residue for the occurring conversion was studied by mutating the cysteine to alanine. No conversion was found using mass spectrometry when the
96:
Aldehydes and ketones are therefore best used in compartments where such unwanted side reactions are decreased. For experiments with life cells , cell surfaces and extracellular space are typical fielding areas. Nevertheless, a feature of carbonyl groups is the vast number of organic reactions that
122:
The formylglycine tag or aldehyde tag is a convenient 6- or 13-amino acids long tag fused to a protein of interest. The 6-mer tag represents the small core consensus sequence and the 13-mer tag the longer full motif. The experiments on the genetically encoded aldehyde tag by
Carrico et al. clearly
113:
Carrico et al. pioneered the insertion of the modified sulfatase motif peptide into proteins of interest in 2007 . Such use of aldehydes and ketones as a chemical reporter in bioorthogonal applications has been applied in self-assembly of cell-lysing drugs , the targeting of proteins , as well as
109:
and was discovered already in the late 1990s . Interestingly, the deficiency of FGE leads to an overall deficiency of functional sulfatases due to a lack of α-formylglycine formation vital for the sulfatases to perform their function. FGE is essential for protein modification and need of high
192:, the engineered aldehyde tag consists of six amino acids. A set of organisms from all domains of life was chosen and the sequence homology of the sulfatase motif was determined. The sequence used is the best consensus for sequences found in bacteria, archaea, worms and higher vertebrates. .
558:
Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R. and
Rudolph, M. G. (2005) Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme. Cell 121,
548:
Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R. and
Rudolph, M. G. (2005) Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme. Cell 121,
398:
Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R. and
Rudolph, M. G. (2005) Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme. Cell 121,
36:(-CHO, an aldehyde) at the α-carbon. The sulfatase motif is the basis for the sequence of the peptide which results in the site-specific conversion of a cysteine to a formylglycine residue. The peptide tag was engineered after studies on FGE recognizable sequences in
175:
The aldehyde tag is genetically inserted into a protein of interest. In this example, the human growth hormone (hGH, PDB:1HUW), one of the four initially examined proteins,is shown. The N-terminus of the protein is fused to the formylglycine aldehyde tag.
123:
showed the high conversion efficiency with only the core consensus sequence present. Four proteins were produced recombinantly in E.coli with a 86 % efficiency of for the full-length motif and >90 % efficiency for the 6-mer determined by
101:
as a ligation. The reaction is known from natural product biosynthetic pathways and has the major advantage that a new carbon-carbon bond is formed. This guarantees long-term stability compared to carbon-heteroatom bonds at same reaction kinetics .
408:
Carrico, I. S., Carlson, B. L. and
Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322. Jencks, W. P. (1959) Studies on the Mechanism of Oxime and Semicarbazone Formation1. J. Am. Chem. Soc. 81,
454:
Hudak, J. E., Barfield, R. M., de Hart, G. W., Grob, P., Nogales, E., Bertozzi, C. R. and Rabuka, D. (2012) Synthesis of
Heterobifunctional Protein Fusions Using Copper-Free Click Chemistry and the Aldehyde Tag. Angew. Chem. Int. Ed. 51,
492:
Dierks, T., Miech, C., Hummerjohann, J., Schmidt, B., Kertesz, M. A. and Figura, K. von. (1998) Posttranslational
Formation of Formylglycine in Prokaryotic Sulfatases by Modification of Either Cysteine or Serine. J. Biol. Chem. 273,
529:
Roeser, D., Preusser-Kunze, A., Schmidt, B., Gasow, K., Wittmann, J. G., Dierks, T., Figura, K. von and
Rudolph, M. G. (2006) A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme. PNAS 103,
97:
involve them as electrophiles. Some of these reactions are readily convertible to ligations for probing aldehydes. A rather exotic reaction recently employed for bioconjugation by
Agarwal et al. is the adaptation of the
482:
Dierks, T., Lecca, M. R., Schmidt, B. and von Figura, K. (1998) Conversion of cysteine to formylglycine in eukaryotic sulfatases occurs by a common mechanism in the endoplasmic reticulum. FEBS Letters 423,
388:
Dierks, T., Lecca, M. R., Schmidt, B. and von Figura, K. (1998) Conversion of cysteine to formylglycine in eukaryotic sulfatases occurs by a common mechanism in the endoplasmic reticulum. FEBS Letters 423,
473:
Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. and Stüber, D. (1988) Genetic
Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Nat Biotech 6, 1321–1325
379:
Miech, C., Dierks, T., Selmer, T., Figura, K. von and Schmidt, B. (1998) Arylsulfatase from Klebsiella pneumoniae Carries a Formylglycine Generated from a Serine. J. Biol. Chem. 273, 4835–4837.
539:
Sase, S., Kakimoto, R. and Goto, K. (2014) Synthesis of a Stable Selenoaldehyde by Self-Catalyzed Thermal Dehydration of a Primary-Alkyl-Substituted Selenenic Acid. Angew. Chem. n/a–n/a.
370:
Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D. and Bertozzi, C. R. (2013) A Pictet-Spengler ligation for protein chemical modification. Proc Natl Acad Sci U S A 110, 46–51.
67:. Since the pH-optimum of 4 to 6 cannot be achieved by adding a catalyst due to associated toxicity, the reaction is slow in live cells. A typical reaction constant is 10 to 10 M s.
436:
Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. and Schultz, P. G. (2003) A New Strategy for the Site-Specific Modification of Proteins in Vivo†. Biochemistry 42, 6735–6746.
334:
Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. and Schultz, P. G. (2003) A New Strategy for the Site-Specific Modification of Proteins in Vivo†. Biochemistry 42, 6735–6746.
180:
The FGE recognizes the motif and the cysteine (Cys) residue is converted into the formylglycine residue . The chemical reporter is formed on location by an enzymatic reaction.
427:
Chen, I., Howarth, M., Lin, W. and Ting, A. Y. (2005) Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Meth 2, 99–104.
445:
Mahal, L. K., Yarema, K. J. and Bertozzi, C. R. (1997) Engineering Chemical Reactivity on Cell Surfaces Through Oligosaccharide Biosynthesis. Science 276, 1125–1128.
361:
Stöckigt, J., Antonchick, A. P., Wu, F. and Waldmann, H. (2011) Die Pictet-Spengler-Reaktion in der Natur und der organischen Chemie. Angew. Chem. 123, 8692–8719.
55:. They form stabilized addition products with carbonyl groups that are favoured under the physiological reaction conditions. At neutral pH, the equilibrium of
418:
Rideout, D. (1994) Self-assembling drugs: a new approach to biochemical modulation in cancer chemotherapy. Cancer Invest. 12, 189–202; discussion 268–269.
47:
due to their strong electrophilic properties. This enables a reaction under mild conditions when using a strong nucleophilic coupling partner. Typically,
145:
has endogenous FGE-activity. The introduction of an aldehyde tag as proposed by Carrico et al. has a workflow that consists of three segments:
520:
Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
511:
Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
502:
Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
464:
Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
325:
Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
307:
Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
110:
specificity and conversion rate is given in the native setting, which makes this reaction interesting for chemical and synthetic biology .
40:
from different organisms. Carrico et al. discovered a high homology in the sulfatase motif in bacteria, archaea as well as eukaryotes .
343:
Lim, R. K. V. and Lin, Q. (2010) Bioorthogonal Chemistry: Recent Progress and Future Directions. Chem Commun (Camb) 46, 1589–1600.
86:
competition with endogenous aldehydes or ketones in metabolites and cofactors. Lower yields and impaired specificity can occur.
213:
intermediate. Subsequently, the hydroxyl group is transferred to the cysteine of the substrate and after hetero-analogous
131:
and has the advantage that it can also be genetically encoded. The sequence is recognized in the ER solely depending on
209:. The key step of the catalytic cycle is the monooxidation of the cysteine residue of the enzyme, forming a reactive
316:
Jencks, W. P. (1959) Studies on the Mechanism of Oxime and Semicarbazone Formation1. J. Am. Chem. Soc. 81, 475–481.
71:
44:
29:
184:
The carbonyl group is probed using typically hydrazide- or alkoxy amine-functionalized dyes or other compounds.
106:
59:
formation, is lying far to the reactant’s side. To make more product named compounds are used to form stable
221:
is formed. This compound is very reactive and easily hydrolyzed, releasing the aldehyde and a molecule of H
98:
82:
in certain cellular environments. Limitations of aldehydes and ketones as chemical reporters are due to
78:
and the herein discussed aldehyde tag , . Limiting the use of aldehydes and ketones is their restricted
275:
is formed. The equilibrium lies far to the product side due to the high reactivity of the thioaldehyde
149:
the expression of the fusion protein, that carries the peptide tag derived from the sulfatase motif,
79:
352:
Prescher, J. A. and Bertozzi, C. R. (2005) Chemistry in living systems. Nat Chem Biol 1, 13–21.
135:
and subsequently targeted by FGE . Notably, in the setup of recombinant expression proteins in
75:
201:
mutated peptide tag was used . The mechanism shows the important role of the redox active
8:
124:
132:
214:
255:
The hydroxyl group is transferred to the substrate and a substrate-sulfenic acid
105:
The modification of cysteine or, more rarely, serine by FGE is a rather unusual
114:
glycans and the preparation of heterobifunctional fusion proteins since then.
52:
164:
89:
side reactions like oxidation or unwanted addition of endogenous nucleophiles.
210:
218:
33:
202:
56:
240:
195:
60:
48:
37:
92:
restrained set of probes that form sufficiently stable products , .
235:
Conversion of Cys to f(Gly) by FGE from Dierks et al. : Substrate
137:
128:
25:
228:
141:
a coexpression of exogenous FGE aids full conversion , although
19:
259:
is generated. β‑elimination of water leads to a thioaldehyde
127:. The size of the sequence is analogous to the commonly used
64:
157:
the bioorthogonal probing with hydrazides or alkoxy amines (
74:
using different techniques, including modern methods like
291:= substrate protein cysteine embedded in sulfatase motif.
117:
205:of cysteine in the formation of f(Gly), as seen in
196:FGE-mechanism of cysteine-formylglycine conversion
70:A carbonyl group is introduced into proteins as a
153:the enzymatic conversion of Cys to f(Gly) and
283:and its arising tendency to form the hydrate
171:: Formylglycine aldehyde tag Carrico et al.:
20:Development of the formylglycine aldehyde tag
32:(FGE). Formylglycine is a glycine with a
227:
163:
251:of FGE is oxidized to a sulfenic acid
118:Genetically encoding the aldehyde tag
13:
43:Aldehydes and ketones find use as
24:The aldehyde tag is an artificial
14:
571:
51:and aminooxy probes are used in
552:
542:
533:
523:
514:
505:
496:
486:
476:
467:
458:
448:
439:
430:
421:
412:
402:
392:
382:
30:formylglycine-generating enzyme
373:
364:
355:
346:
337:
328:
319:
310:
301:
107:posttranslational modification
1:
295:
263:which is quickly hydrated to
7:
10:
576:
267:and after elimination of H
279:compared to the aldehyde
99:Pictet-Spengler-reaction
292:
185:
76:stop codon suppression
28:tag recognized by the
243:isomerization occurs
231:
167:
293:
239:binds to FGE, and
186:
45:chemical reporters
271:S, the aldehyde
125:mass spectrometry
72:chemical reporter
567:
560:
556:
550:
546:
540:
537:
531:
527:
521:
518:
512:
509:
503:
500:
494:
490:
484:
480:
474:
471:
465:
462:
456:
452:
446:
443:
437:
434:
428:
425:
419:
416:
410:
406:
400:
396:
390:
386:
380:
377:
371:
368:
362:
359:
353:
350:
344:
341:
335:
332:
326:
323:
317:
314:
308:
305:
133:primary sequence
80:bioorthogonality
575:
574:
570:
569:
568:
566:
565:
564:
563:
557:
553:
547:
543:
538:
534:
528:
524:
519:
515:
510:
506:
501:
497:
491:
487:
481:
477:
472:
468:
463:
459:
453:
449:
444:
440:
435:
431:
426:
422:
417:
413:
407:
403:
397:
393:
387:
383:
378:
374:
369:
365:
360:
356:
351:
347:
342:
338:
333:
329:
324:
320:
315:
311:
306:
302:
298:
290:
270:
250:
224:
198:
120:
22:
16:
12:
11:
5:
573:
562:
561:
551:
541:
532:
522:
513:
504:
495:
485:
475:
466:
457:
447:
438:
429:
420:
411:
401:
391:
381:
372:
363:
354:
345:
336:
327:
318:
309:
299:
297:
294:
288:
268:
248:
222:
197:
194:
119:
116:
94:
93:
90:
87:
53:bioconjugation
21:
18:
9:
6:
4:
3:
2:
572:
555:
545:
536:
526:
517:
508:
499:
489:
479:
470:
461:
451:
442:
433:
424:
415:
405:
395:
385:
376:
367:
358:
349:
340:
331:
322:
313:
304:
300:
286:
282:
278:
274:
266:
262:
258:
254:
246:
242:
238:
234:
230:
226:
220:
216:
215:β-elimination
212:
211:sulfenic acid
208:
204:
193:
191:
183:
179:
174:
170:
166:
162:
160:
156:
152:
148:
144:
140:
139:
134:
130:
126:
115:
111:
108:
103:
100:
91:
88:
85:
84:
83:
81:
77:
73:
68:
66:
62:
58:
54:
50:
46:
41:
39:
35:
31:
27:
17:
554:
544:
535:
525:
516:
507:
498:
493:25560–25564.
488:
478:
469:
460:
450:
441:
432:
423:
414:
404:
394:
384:
375:
366:
357:
348:
339:
330:
321:
312:
303:
284:
280:
276:
272:
264:
260:
256:
252:
244:
236:
232:
219:thioaldehyde
206:
199:
189:
187:
181:
177:
172:
168:
158:
154:
150:
146:
142:
136:
121:
112:
104:
95:
69:
42:
34:formyl group
23:
15:
203:thiol group
188:As seen in
57:Schiff base
455:4161–4165.
296:References
217:of H2O, a
129:6x His-Tag
61:hydrazones
49:hydrazides
38:sulfatases
241:disulfide
233:Figure 2:
559:541–552.
549:541–552.
409:475–481.
399:541–552.
225:S , , .
169:Figure 1
143:E. coli
138:E. coli
26:peptide
530:81–86.
483:61–65.
389:61–65.
207:Fig. 2
190:Fig. 1
159:Fig. 1
65:oximes
287:. Cys
247:. Cys
289:Subs
63:and
257:III
253:IIc
249:341
245:IIb
161:).
281:VI
277:IV
273:VI
261:IV
285:V
269:2
265:V
237:I
223:2
182:C
178:B
173:A
155:C
151:B
147:A
Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
