Vol. 171, No. 3, 1990 September 28, 1990

SEQUENCE

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1319-1325

SIMILARITIES BETWEEN TRYPTOPHAN SYNTHASE @ SUBUNIT OTHER PYRIDOXAL-PHOSPHATE-DEPENDENT ENZYMES Peer

Academy of Biology,

Sciences Department

Bork

of of

and Klaus

AND

Rohde

the GDR, Central Biomathematics,

Institute Berlin

of 1115,

Molecular Germany

Received August 20, 1990 Summary: On the basis of 8 tryptophan synthase j3 subunits (EC 4.2.1.20) consensus patterns were constructed comprising two conserved motifs. Screening of the SWISSPROT protein sequence database with these patterns indicates similarities with Oacetylserine sulfhydrolases (EC 4.2.99.8), threonine synthases (EC 4.2.99.2), L- and D-serine dehydratases (EC 4.2.1.13/ EC 4.2.1.14) and threonine dehydratases (EC 4.2.1.16). Using multiple alignment procedures the similar regions could be extended. In connection with their pyridoxal-phosphate-bindingcapacity and their positions in biochemical pathways evolutionary relationships among these enzymes are discussed. 01990 Academic Press, Inc.

With the progress in sequencing techniques more and more primary structures of pyridoxal-phosphate-dependent enzymes become available. This offers the chance to get information about tertiary structures, functional mechanisms and evolutionary relationships. Since pyridoxalphosphate is a cofactor of different enzymes like transaminases, decarboxylases, phosphorylases, dehydratases, synthases, sulfhydrolases etc. and since pyridoxal-phosphate is able to catalyse many of the respective reactions alone (without enzymes) the structures and sequences of these proteins can be expected to vary to a great extent and there is no reason for a common ancestor. This is confirmed by high resolution X-ray crystallography of glycogen phosphorylase A and B (1,2) some aspartate aminotransferases (mitochondrial isoenzyme from chicken heart (3), cytosolic isoenzymes from pig (4) and chicken heart (5)) and tryptophan synthase (6) for which no structural homology could be obtained. Other than, for instance, in nucleotide binding proteins (7) the binding sites (around the essential lysine) are different in the published tertiary structures (1,5,6).

1319

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

171,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pyridoxal-phosphate-dependent enzymes are involved in many biochemical pathways, but an accumulation is found in anabolic reactions of the aspartate family and in connected pathways. Some homologies among pyridoxal-phosphate-dependent enzymes of the biosynthetic pathways of the aspartate family could be identified Despite different functions they seem to have similar (8). structures. Recently, distant homologies were found also between some aminotransferases (9), but the enzymes of this family act in a completely different way. As a further application of our simple property pattern which was also sensitive in identifying difsearch method (lo), we propose here a distant ferent nucleotide binding sites (ll), homology between tryptophan synthase p subunit and other pyridoxal-phosphate-dependent enzymes. Homologies among this family of pyridoxal-phosphate-dependent enzymes allow structural and functional insights in the mechanisms of all these enzymes and provide some hints related to the evolution of biochemical pathways. Materials

and Methods

By means of FASTP (12) the sequences most similar to tryptophan synthase p subunit of salmonella typhimurium (with known structure) could be identified in SWISSPROT protein sequence containing 12305 sequences). Significant database (release 12.0, matches were given by tryptophan synthase p subunits from other species and the equivalent part of the uncleaved yeast enzyme. The 8 sequences found in this way were aligned using our program MULTIS (13) which allows to compare up to 15 protein sequences now. In tryptophan synthase pyridoxal-phosphate is covalently bound to an essential lysine of the p subunit (or to the equivalent region of the uncleaved enzymes). A second conserved region is a glycine rich turn which ligates through H-bonds the phosphate group of pyridoxal-phosphate (6). For these regions property patterns were constructed using PAT (14). This procedure leads to rows of amino acids in each position of a sequence segment (fig. lb) . With these patterns the SWISSPROT database was screened. Since our simple property pattern procedure is able to recognize also distantly related proteins (lo), for multiple alignment of protein sequences with a low degree of amino acid identity we have developed an additional program PULIN, which carries out the multiple alignment via an iterated profile alignment (15-17) using the Dayhoff PAM250 matrix (18). We used PULIN to align those proteins, for which some similar regions were detected by our property pattern method. Results When the SWISSPROT was screened comprising the two motifs described

with our property above in addition

pattern to the

Vol.

171, No. 3, 1990

CYSKCECOLI CYSKSSIILTY SDHDSECOLI SDHLSRAT THDlSECOLI THDZSECOLI THDHSYEAST THRCSBACSU THRCSBRELA THRCSECOLI TRPSYEClST TRPBSBACSU TRPBSBRELfi TRPBSCAUCR TRPBSECOLI TRPBSPSEPE TRPBSPSEPU TRPBSSALTY b)

BIOCHEMICAL

0-acetylserine

sulfhydrolase

D-swine L-swine threonine

dehydratase dehydratase dehydratase

threonine

synthase

tryptophan tryptophan

synthase synthase

25 25 100 24 45 41 92 42 42 90 367 73 BZ 82 69 76 78 69

0 subunit

:

corresponding

allowed

amino

tryptophan binding

synthases sites

(7)

3 3 2 1 0 0 0 1 0 2 0 0 1 0 0 0 0 0

RESEARCH COMMUNICATIONS

ffiRIL@KvESRNP-SFSvKCRiGIlN NGRILAKvESRNP-SFSvKCRiGAN SGQLLLKKDSHLPISGSIKaRGGIy GTSVFLKMDSSQP-SGSFKIRGIGh DNVILVKREDRQP-VHSFKLRGAYPl KGEIFLKFENMQR-TGSFKIRGOFN NTNVILKREDLLP-VFSFKLRGAYN GIELHVKtEGVNP-TGSFKDRGMVM GVQLYGKYEGCINP-TGSFKDRGIIVI’I ESDVGClElFHGP-TLAFKDFGGRF GAQIWLKREDLNH-TGSHKINNClLA GAKIYLKREDLNH-TGSHKIMLG f&RIFLKREDLVH-GGAHKTNQVIG GAKIYFKRDELNH-TGSHKINNALG NTTLYLKREDLLH-GGAHKTNQVLG GAKIYLKREELNH-TGCIHKINNCIG GAKIFFKREELNH-TGAHKVNNCIG RTTLYLKREDLLH-GGAHKTNQVLG

167 167 269 194 178 174 228 175 175 238 520 225 234 234 221 228 230 221‘

3 3 1 0 1 0 0 0 0 3 1 0 1 0 0 0 0 0

CIAAI~CIKED~~ACIAII~FKCCAACIA CCCLCC FECCCF-CCSH DGCCCC DGDVFF H DFGH GF I EHGGFF EIE GG K EHIK NG K IKMIGG GLI HI II FILM SH L LllNLHll KMK IL 0 GKMN T I II MNQtiIN IINL KM R ULNP VK W NQSVKQ NON LV W NMQQ L Y QRT LS QSN 11 Y QNSR M T T MT RTQ T SQTT T VW NW SVR V TRVW V Y QY R TSW WSY w TY YT Y S V T V W V Y W Y

Lysine-motif and phosphate-dependent

1.

Fig.

acids:

AND BIOPHYSICAL

also of

glycine rich enzymes.

the

turn

proposed

2 threonine

GQVDVfIAqVGTGGTLTGVsRYI GQVDVfIsgVGTGGTLTGVTRYI DNPLFVYLPCGvGGGPGGVAFGL AKPGAIVLSVGGGGLLCGVVQGL AHLDRVFVPVGGGGLAClGVAVlI YDVDNVIVPIGGGGLI~GIAVAI NKIGPlVFVPVGGGGLIAGIGGYL EPPDVLAIPVGNAGNITAYUKGF WPDVLAIPVGN&GNITAYUKGF RNQLVVSVPsGNfGDLTAGLLAk kLPDAVVACVGGGSNSTGnFSPF TflPDKVVACVGGGSN4I’lGHFQAF kLPDVVVACVGGGSNAIG?lFPlDF RLPDAVVACIGGGSNAIGLFHPF RLPDAVIACVGGGSNAIGNFfiDF RLPDSLVlrCIGGGSNAMGLFHPF RLPDSLVACVGGGSNAtlGLFHEF RLPDAVIACVGGGSNAIGtlF&DF CICICAACCIIIACGCIA~~~4~~~~~F CCICCFCCCI CGGCCCGCCCCI DDLDFIFGNL G SDIG FFDDL EEMEHLIIPI’I N ELI GGEEM FFNGIMLLSV S GIIL IIFF GGPIKVHMT T IM LLHG HHQLL NV LPT MflIM KITMM Q tlQV TTKN MKVNN S NS VVLP Q-f WWNQ Nt 00 T Qfl SR V SV YYQS RN TS II T RT SO VT Y V SW TR V TY ws II V YT Y W V Y

of pyridoxal-

pyridoxal-phosphate

dehydratases,

2 threonine

synthases and a serine dehydratase match the pattern with only a few deviations. The respective regions of the detected enzymes were used to screened threonine

improve our property With this second again.

pattern run all

and the SWISSPROT was threonine synthases,

dehydratases and serine dehydratases of the SWISSPROT In addition 2 0-acetylserine sulfhydrolases were found.

were detected

for

tryptophan the results first

which

synthase of this

and up to

similarities

of

their

C-terminal

part

to

p subunit were described (19). Fig.la shows run. Up to 5 mismatches (deviations) from the

4 from

the

second

motif

were allowed,

but

out

12305 proteins in.SWISSPROT only the proteins shown in fig.la With a third run on the bases of property match both motifs. patterns derived from all regions shown in fig.1, no further sequence segment was detected before 8'noise1' appeared. 1321

of

Vol. 171, No. 3, 1990

a. THD1lECDLI 0 lNDZ#ECDLI 0 lM#l#VEAST 31 THRcIBAcBu 0 lHRC#SRELA 0 THRC#ECULI 25 0 sdhI#hhfihn 0 SOHL#RRl SHDD(ECOLI Jz TFiPB4SALlV I TRPBIPSEPU 9 rRPB4cALfCR I2 TRPB4BRELn 10 TRPBIBACSU 4 IRPIVEnST 0 CVSX4ECOLI 0 CVSKISALTV 0

THDZ#ECOLI 82 THDI4ECOLI 36 JHDH#VEAST 133 THRCo(IC5U 52 THRClDRELn 83 THRC(ECOII bdhllhdd6h 64 SDHLIYT 67 SDHD(TCDLI 145 lRPB4SnLTv 110 lRPB4PSEPU 119 TRPSICnUCR 123 TRP54BRELR TRPB45nCSD 114 lRP#VEntT 109 CVSK4ECOLI 66 CvSKwlLTv 66

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

4 445 434 33 3335.4. 34 345.!34.3..33 4 3 4 4 .. s TPL L L KE P 55KR A UK ----------------------------IITYDLWAIWI5-------------------------~D~5~~~~~~~E~W~~K~----~lL~~-~~~V~l~5K~IlA5--___ NTKAlRHROH5FSLIKLH____ SELKLDflPTPILIPOVVRL___ -8BR) ---------------------------~KSLl~KEFLPVl~lP~~~~##l~PK#5EE~---5IEL~l~-l#~#~~#~E~Tl~~----------------------------~VK~LLKOVA~VLPVNEKlPDVU~~~##IPLLN1~K~#----tV~Vt#V#t~-l#~~~~#~~AlI~Y--___ EStLFFPHDLPEF5LlEID _-__ ERLKLDFVTMAKIL5AFIt__ DEIWANV __ ___-___ ___ _ _---- __-__-_-__ ___--- _-___ -----A5tE~~-KHIRD~tK~----tT~VLM~W-~~~#l~l~C~K~V-~-__ __ _. - - -- _ ___ - _- _ ___ ___ -_ __ __ __- -----------NA~#LHV-K###R~A#tKVA---~l5~L~D5~#-5#~#I~I~CKNKAK~~V-~ ____ Sp TTTLAE5LPVVt~5RFAPVLAKAF~V55Pl 1 TlLLYVF5EF66llVWPILHP~N~EEAFVRllelDPlTA8T---RTTLVLWDUH-WIYWT~L#~K~ll~TS-tPDnfflLF~Ft6RVVnETLllPLVLDLAREYElAXAWIFKIFF#~EL~-l#~l~~L#K~K~LI#TB-VPDAEtRF~tFt~VVnETLRPLVLDLtKnVADAKADPEF#ARKtVN~HV~R~##VF~R#TEHF---t~AKIVF#R~L~-l#~~~~L~lU#~KTR~I~Tt-STLLPnVFGEFtS5FVIIESLLPnL~LEKAFVDAT~~F~ELWVLRDVLt~###TEflN#PLAtEtK~AR~FL~~~-t~l~l~L#K~KlRIl~Tt-YPNEItRVtDF~~KFVPElLH~LDEI5TnFKPIKDDPAFREEVVKLLKDV~~~#lVA~TEV#---WAKlVL#R~~~-l~#I~~~KK~KT#lI~T~--v --KHPIRFSDFGBPVVPEALHACLRELEKtFDEAVADPTFNEDFK~~TE~---~ ____ _- - __ _____-_____ __ ________ ------------------N#KIFE~S#ll~TPLVRII__ __- ---- ___ __. - -__ __ _- -_ ____ _ __--_____ __ --____- R#KIVEDRS#TI5HTPLVRlNRI555RlLA#V#5RN#-5F#WC#I5A5NlND#EKR#VL#F5VELV-hhhhhhhhhhhhhh hhhhhhhhhhhhh bbbbb tttt bbbbbbb t t hhhhhhhhhhh bbbbbbb

52 56 52 53 64 66 110 119 123 123 114 66 66

3!4.4 4.5 .455545.1.44.4 4.3 .3 33331. 33 Sk, 3N 3 A A AnARLt K T I R P A A ---------------------------------A#~~~-LtC~##l~KVV#---------#Kt~K---5KV~TCD----------V~~~TIAK~l~ 143 ---------------------------------A~~l-F~####V#AL#V#---------#TATAD---lKV~~t---------~~~~~#K~lEL~ 147 _________________________________ vAl#V# ---------------------------------T##T5A#A#-AV###~#ClVII---------~KIAF--BKL~----------VBIlIAII(#FH(CKI~iEE 144 ________________-________________ T~l3n5Y-AV##H#)I1CI#VI---------#EtK~AH-~KL~~----------V~~I5IE~~KA~l#A 144 ______--________--______________ T __--_____ -----____---------------------------------A##A#~A#-V##R##~M#VV---------#tllPALTIE~K~~TCK------~~LLDE~E~K~A~ 151 5IYGSRIRGAtHtDEOPHVRtDALLPDT~PLTA##A#H#T#-V##R~#LPA##VV---------#tTTPALTlER~~TVE-------~~~Al~AK#LEK~ 166 DDV5---------KLLSPEFK5FFS5V5InV551#~ --____-----___----_-_____________ AMHW#t#-L#S#L##LKR#V#~WE~~R~~~VI~~lLK-D~~M~tVET~V~tT~ 192 -________---_______--.----------A#HH#V#T#-TV###F#LPCV#V#~lDlE~#N~RRKLLtAEl~T~T5TLK-~~~TN~DlFVLI5TW5 201 ---------------------------------A#5~V#l~-TVC#~F#LP~VV#~nTD~~K~~~LtAEV~~~ltTLK-~~~~DTVVLltTAW 205 _________________________________ A#OH#T#T#-L#C#LII#LECVVV#~KDVAR5##NVV~H~KVI~tt~lLK-~~~R~TATF~~VLLtT~ 205 ---------------------------------A#QH#V#P#-TV##KF#FSC#VFWEEDVn~SLN~~KLL~~~T~TLK-~T~AIRV~~~~It~ 196 ---------------------------------A#~#V#T#-l#C#KF#LlC#~#5~D~W~TKlLR-~T~TTVVV~l5 IeL ------------------------------EPlS##l#I#L#VVH##-#V#L#LT#------PETIIBIEIRnLXKBM IS2 ------------------------------EPTN##l#I#L#VV####-#V#L#LT#------PET~IERRKLLK~~V ---------JlE(MK----8lOK~IVA 152 hhhhhhhhhhht t bbbbbb hhhhhhtt bbbbbb h hhhhhhhhhhht tt bbbbb

123

1HDZlECOLI 144 lHDl#ECOLI 145 1HDNlVEAST 195 THRClSACSU I45 THRC4BRELA 145 lHRC#ECOLI I99 sdhl4hudm 132 SBHLIRAT 167 SDHD4ECOlI 150 TRPS45ALTY 193 IRPMPSEPU 202 7RPBtcnLicR 206 TRPB4URELn 206 TRPB4BnCSU 197 cR@TEj@L 192 CVSKIECOLI 133 CfSI4SALTV 153

0.

&

THD24ECOLI 245 THDl#ECLUI 247 TH~VE~T lHRC#SK5N 24s lHRC##RELn 243 1HRClECOLI 306 sdhl4hdhm 231 SDULIRRT 264 5DtlMECMI 344 TRFl45ALlV 297 lRPB#PSEPU 306 TRPBlCAUCR 310 TRPBlBRELn 310 TRPBIBACSU 300 TRPlVEASl 249 CVSK#ECMI 249 CVSKISnLTV 167

4 3 34 334..3 4 . 34343..5!344.3543 .43 3 3.43 .3. 443 545. 4 33. 433 3. P P 3 n DV VtSS 5 K I Dt A 3 A IIEt------RIFIPPVDD#KVIA#~TltLEl~k----VDV#N#I~I~##LlA#IAVAI#5I~lI~W---V5~~~~-----llT~TTtTL#~~~ 242 ~~~------FT~~FDH#~~A#~TL#LELLWD----nHL#R~VP~~~~L~~VL~~~lKV#A---~~~~K#M~----~~P~F~~A~ 246 ____-,, ---__ T ER5 LT IPPFDHIVVIA##STV~IKT#5---VElVmN5 R PLPW5 TF#DaTgVRll KS#-------InLVN~~VRIE#OKln#FEVCEPL---~~#~AIWt~#NITAVNK~#EV~K~~tL-PK~tFEA~~~I~-----V~E~--Tl~TA~R~~ 242 EE#-------ITLVfflVW#VRlE#OKTA#FElCDRL---W~lVLAIP#~#NIlAVNK5FCEVEKEK5VKK-~l~EA-E~#AlVK~-----VlEEPE--lI#TAIRl~ 242 DEE-LKV~5LN~A~P5ETRN5LV#tVP~~F#DL~ntLLAK~L~PVK~#A--Al~~lV~F~~~~-------P~T~AT~ VI#-----------PFDD#LINE#HASIVKELKElL---~KPtAIALS#~##LLC#VVE~~~D~VI--n~TFt~tFO#AV~#KL~LR~LPK~T--~#K~t~ 230 VIS-----------PFDD#LlNE#HltLVKELKElL---5AKPtnIVLS~##LLC#VV~L~~DVP#I--A~TF5~~~~KE#KL~RTLPKIT--~#K~~T 263 SD#NCFF~tRTLFLtVtVR#5RLK#~AO~RIV~DN~F#VLPCtV##SPt#VAF~~~~ --F AEPTH#PC-T PH#--------VPlIVREF~~I#EETK#PILDKE----tRLP#n#InC#6t#t~~#~A~INDTtV~l~E~~lEl~~~-~~VF~K~T#~lEE5 296 PH#--------VP~RDFPSII#KElR#5L~KE----5RLP#SLV~~5#~A~LFHEFLEEP~#IIt~~~~TDK~~L~-~lVLL#~~lT~ 305 PH#--------VPVIIVRDFOSVI#AE~EUILEHE----~LP#n#VACIt~~nI~F~ffLt~~It~AA~TDK~~T~-~~N~lVLLMD~#ll~ SO9 PH#--------FPIIWIEFHKVISEEnK#QALERT----BKLP#V#VnC#~#~AI#NFADFlD~5~L~PA~~#M~lTIl~E-I5lL~T~L~~t 309 PH#--------VP~VVREFOKRl~EAKD~LKRIE----tT~#K~AC#tt#SN~~t~L~D-~LI~~K~I#lPLNA~TItK#T-~I~LTVLI~~F~IIE? 299 PH#--------VPTLVRlF5SVl#KETKE#FAnNN---N5KLPl AIVY(B818WBTHIFSPfEHDTBVolwF SN~----EKVLLL~PF~N#ANPEl~KlTtPE~~D--TDt~V#VFI~Wl~#TLT#~VI#tTK-tKlL~-VA~PT##P-----------------------VIM~~ 218 SD#----OKVLLL~SN#AEI~KT~tPEI~D--~D5OV#VFltt#tT##lLT~VlRV~M~K-~KlL#T-VAV~T##P-----------------------VI~~ 215 bbbbbbtthhhhhhhhhhhhhhh bbbbbbb hhhhhhhhh bbbbbbb 4 3 33.4 333355. 4.5 53434433 .433.535454 4 3 34.435351.4.4 L V E A LEE! E AALA5L NTV L58 Pt-----NLTV--------EIVRELVDDl~~D#I~IA#l~KW--Tl~~~#~#~KL~~----------~RKT#51l~~IM~--VWlTffV~Il-----DElF-------WCPEVLWIITV)BIMICIIUIIDH)WRIW--MPBBK#HM~5~4 16_____ EETF________ myIIWWRWIVIIWIWNYXDIF(DTR319NPE-_ -_-___PfflN---DKAV--------KA~E~KI~V~MILH#V~I~#~--A~~l~#K~K~l---------~WK~V#T~K~T~I~IK~~~SSZ PIIBN---tVnV--------EA~~1UWTWICA~KDIF#Dl~I--A~~IK~8BKI----------KK~W~l#~K~-lAl~DI~~352 F------W PNNNPRVEEL I(OCXELBYMIVDMTT~~TRRE#-KEL#~T-S~FL~T~AKFKE~~KELAERA-~ VS-----PTL--------K#FVEHPIF5EVlS~#~A#lEKV-DDlKIL--V#P~t~~V~l#KLO----LE~l~~~l~I~~~K~~l~~S~ VG-----nOTL--------K#FVS~lFSEVl5~#~T#IEKFVDD#KIL--WPAC~#~AVV~~~5----~~L#T~~#VI~~I~~~K~~~~~~ A~-----~Vt--------RANERL~LtWTNVD~t~##I~-L#P~-#L~#~~A~V~ _-__ VSISA6-LDFPSVt-PPHAV#NSltRADV#5ITDD#ALE~KT~~##lIPAL#88H#L#HHK~BPE------------KE5LL#W#5#R#MIFTVN#lLKM5EI-NSISAt-LDVP81#-PEHAV#~VKR~V~lTD~LD#FHATC~#lIIP~#5~l~#l~~K~--------------K~~~~~K~ HSISAt-LDVPt16-PEHSFHmItR~V#BTTDT#IILE#FKLCSlL##lIPK#P~K#WWI~L5--------------~Kl~D~IFT~~I---VBISA6-LDV~V6HtlHTCT~A-RTTt)SPlPKPSK~-~~#~V##Il~TtlL~~K~Kl~~~----------~~IL~~~~~LI~~4l6 VSISAt-LDVPBIt-PEHAV#HK~~lVD5lT~#AVD#LKL#~K##lLPAl#~~#K~K#AK~D-------------~IL~#K~~ HSVSAS-LDVPWS-PELAVNKtl5RA5FlA PTNK _____________ IKP5~K~D5I~-FIPAN#~KLVDKVI5~~~~AItT~~E~~lL~l~#W~K~~5-------------FT~~~~l~~5T~~~E~5 IXP6PHKI06l8A6-FIPBII(MKLiDKV#6llNE#AISlARR#NIW)MLAtl~~#W~K~~~------------FT#KNl~l~VL~~A~~K~5 hhhhhhhhh bbbbbb hhhhhhhhhhhhh hhhhhhhhhhhbhhhhh bbbbbbb hhhhhhhhhbhhhh

329

297

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396 405 4#6 400 323 323

Vol. 171, No. 3, 1990

Fig.

3.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Metabolic map of phosphate-dependent

amino acids enzymes.

and

pyridoxal-

To verify the indicated similarities among the detected enzymes we have used our multiple alignment program PULIN. The similar regions could be extended and we propose now an alignment, with reveals a similar folding topology in long segments of the included enzymes (fig.2). The similar sequence segments agree with structural findings, because deletions and insertions mostly occur in regions between corresponding secondary structure elements of tryptophan synthase (fig.2).. Our alignment with 0-acetylserine sulfhydrolase is somewhat different to that proposed by Levy and Danchin (19). So we have related the potential pyridoxal-phosphate-binding site in 0-acetylserine sulfhydrolase to a region in tryptophan synthase around.the lysine-87 (numbering according to the enzyme from salmonella typhimurium, fig.2) as indicated by our property patterns (fig-l). Only in 3 positions amino acids were found to be absolutely mark in conserved in all sequences (marked by an exclamation fig.2). One of them is the essential lysine covalently bound to the second is a glycine of the second motif pyridoxal-phosphate, used for the pattern construction and the third is located in a Fig.

2.

Conserved dependent

sequences enzymes.

among

1323

pyridoxal-phosphate-

Vol.

171,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

region surprisingly found to be the most conserved one corresponding to residues 112 to 134 in tryptophan synthase from This region comprises an a-helix salmonella thyphimurium (fig.2). with a subsequent turn and consists of a cluster of tiny amino Further conserved regions comprise the motifs used for acids. pattern construction. All three most conserved regions are not located in the P-sheet core so that they seem to have essential functions in pyridoxal-phosphate-binding. Discussion

The pairwise identities between enzymes of the different subgroups in fig.2 range between 15% and 19%. These values are typical for the so called 'twilight zone' in which usually automatic alignment procedures fail (20,21), but background information argues in favour of their similarities. All enzymes involved in our alignment and proposed to be homologous have some features in common: (i) they all need pyridoxal-phosphate as a cofactor, (ii) there are some similarities in their reaction mechanisms via an intermediate and (iii) they all catalyse successive steps in biochemical pathways (fig.3). The third point led to the hypothesis of an evolution of these pathways from an ancestor pyridoxal-phosphate-binding protein. In fig.3 some successive reactions of the aspartate family and their connections to tryptophan synthase are shown. All of them are catalysed by pyridoxalphosphate-dependent enzymes. Therefore we have also investigated other enzymes of this scheme (fig.3) not found by our pattern and alignment procedures. Homologies between cystathionine r-synthase (EC 4.2.99.9) and cystathionine 8-lyase (4.4.1.8) were detected (22) and we have also found a high level of similarity to 0-acetylhomoserine thiolyase (EC 4.2.99.10). Oacetylhomoserine thiolyase can catalyse the same reaction as Oacetylserine sulfhydrolase which is, in turn, homologous to tryptophan synthase. Despite an amino acid identity of 11% (10 gaps were introduced by PULIN, data not shown) a homology could not be verified. The identical positions are in contrast to the alignment of 0-acetylhomoserine thiolyase with cystathionine P-lyase and cystathionine r-synthase. Nevertheless, the evolution of these pathways can be explained as a series of gene duplication events. In various aminotransferases or decarboxylases only the region responsible for the recognition of the respective amino acid had to be changed 1324

Vol. 171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

after gene duplication to explain the different positions in the pathways. In contrast, the family of similar pyridoxal-phosphatedependent enzymes discussed here reveals very early gene duplication events leading to the development of biochemical pathways. The suggested homology between tryptophan synthase (p subunit) and the other enzymes presented here can be used to build topology models of all these proteins. The conserved regions and positions show that despite the expected variability of pyridoxal-'phosphate-dependent enzymes (see introduction) there seems to exist only a limited set of protein topologies compatible with pyridoxal-phosphate-binding via lysine. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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