Chapter 5
Three-Dimensional Structure of an Endochitinase from Barley Downloaded via CALIFORNIA INST OF TECHNOLOGY on July 14, 2018 at 14:21:59 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Jon D. Robert us and John Hart Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712
Endochitinases hydrolyze chitin, cleaving glycosidic bonds within the insoluble polymer. Chitin is extremely abundant, being found in the exoskeletons of many insects, Crustacea and fungi. Chitinases are of commercial interest since they can be used to create chitin oligomers useful in the chemical, pharmaceutical, and food industries. We have determined the X-ray structure of the chitinase from barley. The model has been refined to high accuracy against 1.8 data. This chitinase has 10α-helicescomprising 47% of the structure. It has a very pronounced cleft, assumed to be the chitin binding and catalytic site. A number of residues, which are conserved within the chitinase family, cluster in this cleft Presumably they play key roles in chitin binding and hydrolysis. Barley chitinase reveals an ancient resemblance to lysozyme; they share a common central core despite lacking any obvious amino acid sequence similarity. In barley chitinase, Glu 67 appears to act as the proton donor and Glu 89 as the general base in the catalytic mechanism.
Chitin has been called the second most common organic molecule in the world, after cellulose. T i e annual production of this renewable biosource has been estimated at between 10 and 100 billion tons (I). The polymer is found in the exoskeletons of insects and crustaceans like crabs and lobsters and is also a major component of many fungal cell walls. Chitin has a number of clinical and industrial uses. It is used in paper processing and the food and cosmetic industries. Chitosan, derivedfromchitin by acid hydrolysis, is being used in water treatment and in burn dressings. Oligomers of chitin and chitosan can act as bioactivators. They induce defense responses in higher plants and exhibit some antitumor effects
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© 1995 American Chemical Society
Saddler and Penner; Enzymatic Degradation of Insoluble Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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ENZYMATIC DEGRADATION OF INSOLUBLE CARBOHYDRATES
in animals. A wide range of uses for the two polymers is reviewed in reference (2), the proceedings of the 5th International Conference on Chitin and Chitosan. Chemically, chitin is a linear, non-branched polymer of p-1-4 linked units of N-acetylglucosamine (NAG). Figure 1 shows a segment of the polymer. Hydrogen bonds donated from the C3 hydroxyl to OS in the pyranose ring add greatly to the tensile strength of the molecule. Chitin is a close relative of cellulose, which has a hydroxyl at C2 in place of the acetylated nitrogen. Also related to chitin is chitosan, which resultsfromhydrolysis of the acetyl group. The presence of the this amine group causes chitosan to be protonated and behave as a poly cation. Chitinases hydrolyze chitin. Exochitinases remove N A G units from the non-reducing end of the polymer. This class of enzyme is fairly common in bacteria but occurs only rarely in higher plants. Far more common are endochitinase which cleave chitin internally. These are found in higher plants, fungi, and bacteria. A few endochitinases also exhibit weak lysozyme activity. That is, they are able to cleave the p-1-4 bond between the N-acetyl muramic acid and N A G found in peptidoglycan. (Conversely, some lysozymes exhibit chitinase activity.) Endochitinases are generally monomelic proteins between 25 and 40 KDa. Four classes have been proposed based on amino acid sequences (3,4). Figure 2 shows a sequence alignment of several representative chitinases from classes I, II, and IV. These classes have homologous catalytic domains, of about 26 KDa. This domain contains the chitin substrate binding and catalytic functions. Class II enzymes, like that from barley, contain only this catalytic domain. Class IV enzymes have three deletions within this main unit, but clearly belong to the same family (4). Class I and IV endochitinases also have an N-terminal cysteine-rich domain of about 50 residues. It is homologous to wheat germ agglutinin (WGA), and presumably binds the enzyme to chitin, holding it to the insoluble polymer. The N-terminal domain is linked to the chitinase domain by a glycine/proline-rich hinge segment Class III chitinases show no apparent sequence similarity to enzymes in class I, II, or IV. They are relatively rare in higher plants, but common in fiingi. Plants have no immune system and have evolved other methods to defend themselves against pathogens such as viruses, bacteria, and fungi. Plants under attack can induce genes which express a battery of defense proteins, known as pathogenesis-related (PR) proteins (5,6). These includeribosomeinactivating proteins (RIPs) (7), pore-forming polypeptides that insert themselves into fungal cells (8), and lytic enzymes such as p-1,3 glucanases and chitinases (9,10). In fact these proteins often work synergisticaUy within a single host This is true in barley (Hordeum vulgare) where three antifungal activities from a RIP, endoglucanase and chitinase have been found (21). Our laboratory has been examining the structure and action of representatives of many of these plant defense proteins. We have solved the structure of plant RIPs including ricin (12) and PAP (13), and crystallized the anti-
Saddler and Penner; Enzymatic Degradation of Insoluble Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
5. ROBERTUS & HART
Three-Dimensional Structure of an Endochitinase
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Figure 1: The molecular structure of the chitin polymer.
1 BARLEY TOBACCO POTATO ARABIDOPSIS RICE POPLAR PEA CORN
20
. . • •• • -SVSSIVSRAQFDRMLLHRNDGAC E Q-CGKQACK^CPSC^CCSNFGWCGNTQDYCGPGKCQ A 0>4CGSOGGGKACASGQCCSKFGWCGNTNDYCGSGNCQS-QCPGG GPGPGPG-GDLGSAISNSMFDQMLKHRNENSC A EOCGRQAGGALCPNGLCCSEFGIICGNTEPYCKQPGCQS-OCTPGCTP— PGP-TGDLSGI ISSSQ.FDDMLKHRNDAAC AVRGEOCGSQAGGALCPNCLCCSQYGWCGSTSDYCGAG-CQS-QCSGGCGGGPTPPSSG GGSGVASIIS PS LFDQMLLHRNDQAC A OCGSQAGNATCPNDLCCSSGGYCGLTVAYCCAG-CVS-QCRN CFFTESMFEQMLPNRNNDSC A EQCGSQAGGAVCPNGLCCSKFGFCGSTDPYCGDG-CQS-QCKSSPTPT 1PT PSTGGGDVGR LV PSS LFDQMLKY RNDGRC A QNCG CQPNFCCSKFGYCGTTDAYCGDG-CQSGPCRSGGGGGGGGGGGGGGSGGANVAhlVVTDAFFNGI-KNCJAGSGC
ll
BARLEY TOBACCO POTATO ARABIDOPSIS RICE POPLAR PEA CORN
40 60 80 100 QAKGFYTYDAFVAAAAAFPGFGTTGSADAQKREVAAFLAQTSHETTGGWA QGKSFYTYNAFITAARSFRGFGTTGDTTRRKREVAAFFACrrSHETTGGirDTAPDGRYAWGYCYLREQ-GWPPSYC^ O^KNFYSYNAFINAARSFPGFGTSGDINARKREIAAFFAOTSHETTGGWASAPDGPYAWGYCFLRER-GWPGDYCPPSS-O^PCAPGR PARGFYTYNAFITAAKSFPGFGTTGDTATRKKEVAAFFGO/TSHETTGGWATAPDGPYSWGYCFKQEQ-NPASDYCEPSA-TlfPCASGE RAKGFYTYDAfVAAANAYPDFATTRDADTCKREVAAFLAQTSHETTGGIfPTAPDGPYS PGKGFYTYDAYFVATEFYPGFGKTGDDDTRKREIJVAFFAOTSQETSGRSIIGEDAPFTWGYCLVNELN-PNSDYCDPKT-KSSYPCVA AGHGFYTYDAFIAAARSFT4GFGTTGDDNTKKKELAAFLAOTSHETTGGWPTAPDGPYAWGYCFVSEQ-hrTQEVYCSPK--D^ EGKNFYTRSAFLSAVNAYPGFAHGGTEVEGKREIAAFFAHVTHETGH •-FCYISEIN-KSNAYCDASNRQtf PCAAGQ.
BARLEY TOBACCO POTATO ARABIDOPSIS RICE POPLAR PEA CORN
120 140 160 180 RYYGRGPIQLSHNYNYGPAGRAIGVDLI^PDLVATDATVGFIO'AIMFIIMTAQPP-KPSSHAVIAGO^SPSGADRAAGRVPGFGVITN KYYGRGPIQISYNYNYGPCGRAIGQNLLNNPDLVATNAWSFKSAIWFWMTAQSP-KPSCHDVITGRWTPSAADRAANRLPGYGVITN KYFGRGPIQISHNYNYGPCGRAIGVDLLNNPDLVATDPVISFKTALWFWMTPQSP-KPSCHDVIIGRWNPSSADRAANRLPGFGVITN RYYGRGPMQLSWNYNYGLCGRAIGVDLLNNPDLVANDAVIAFKAAIWFWKTAQPP-KPSCHAV^ KYYGRGPIQITYNYNYGR-GAGIGSDLLNNPDLVASDA-VSFKTAFWFWMTPQSP-KPSCHAVITGQWTPSADDOAAGRVPGYGEITN DYYGRGPLQLRKNYNYGECGNYLGQNLLDEPEKVATDPVLSFEAAl.WFWMNPHSTGAPSCHEVITGE1fSPSEADIEAGRKPGFGMLTN KYYGRGPIQLTHNYNYGIAGgAIKEDLINNPDLLSTNPTVSFKTAIWFWMTPOAN-KPSSHDVITGRWTPSAADSSAGRVPGYGVITN KYYGRGPLQISWNYNYGPAGRDIGFNGLADPNRVAQDAVIAFKTALWFWMNNVHGVMP -QGFGATIR
BARLEY TOBACCO POTATO ARABIDOPSI RICE POPLAR PEA CORN
200 220 240 IINGGIECGHGQDSRVADRIGFYKRYCDILGVGYGNNLDCYSQRPFA 11NGGLECGHGSDARVQDRIGFYRRYCSILGVSPGDNIDCGNQKS FNSGLLLETM 11NGGLECGRGTDNRVQDRIGFY RRYCSILGVTPGDNLDCVNQRWFGNALLVDTL IINGGLECGRGQDGRVADRIGFYQRYCNIFGVNPGGNLDCYNQRSFVNGLL-EAA IINGGVECGHGADDKVADRIGFYKRYCDMLGVSYGDNLDCYNQRPYPPS IITNGGECTKDGKTRQQNRIDYYLRYCDMLQVDPGDNLYCDNQETFEDNGLLKMVGTM IINGGIECGHGQDNRVDDRVGFYKRYCQIFGVDPGGNLDCNNQRSFA AINGALECNGNNPAQMNARVGYYKQYCQQLRVDPGP
Chitinase
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