Protein sequence analysis of SiaT spi

S. piezotolerans is a marine microorganism living in the deepsea.[226] The genomic DNA

sequence of S. piezotolerans has been sequenced[201] and indexed in Genbank (Access No. CP000472). Among its whole genome sequence, an open reading frame has been identified

for which the corresponding protein expression product has high homology with α/β-2,3 and α-2,6SiaTs belonging to the GT 80 family (www.cazy.org) (Sequence 11.1). From the protein sequence alignment, it is shown that the protein is more similar to 2,3SiaT than to 2,6SiaT. For example, it has 27.9% identity and 42.9% consensus with 2,3SiaTpph, but only 19.3%

identity and 31.4% consensus with 2,6SiaTple. From the sequence feature, it is equipped with

both the typical CMP and lactose binding domains, which are also very similar to those of 2,3SiaTpph. Thus, it is assumed that SiaTspi possibly has a 2,3SiaT function.

1 75 SiaTple (1) MKRIFCLVSAILLSACNDNQNTVDVVVSTVNDNVIENNTYQVKPIDTPTTFDSYSWIQTCGTPILKDDEKYSLSF SiaTpph (1) ---MF SiaTspi (1) --- SiaTspi-U (1) --- 76 150 SiaTple (76) DFVAPELDQDEKFCFEFTGDVDGKRYVTQTNLTVVAPTLEVYVDHASLPSLQQLMKIIQQKNEYSQNERFISWGR

SiaTpph (3) VFCKKIFFLIFISLMILGGCNSDSKHNNSDGNITKNKTIEVYVDRATLPTIQQMTQIINEN---SNNKKLISWSR

SiaTspi (1) ---MLVNNQ---SHNPKLICWQR

SiaTspi-U (1) ---MVASSSSNAQQSPYALEIYIDKATLPSVQQAAMLVNNQ---SHNPKLICWQR

151 225 SiaTple (151) IRLTEDNAEKLNAHIYPLAGNNTSQELVDAVIDYADSKNRLNLELNTNTGHSFRNIAPILRATSSKNNILISNIN SiaTpph (75) YPINDETLLESINGSFFKN----RPELIKSLDSMILTNEIKKVIINGNT-LWAVDVVNIIKSIEALGKKTEIELN SiaTspi (18) HPVNDEALLQGINAASFVS----IASLCQHAATLLAGHPHSHITIYGNT-YWSKDLARLIRYLTRISGVEIKKLE SiaTspi-U (50) HPVNDEALLQGINAASFVS----IASLCQHAATLLAGHPHSHITIYGNT-YWSKDLARLIRYLTRISGVEIKKLE

226 300 SiaTple (226) LYDDGSAEYVSLYNWKDTDNKSQ--KLSDSFLVLKDYLNG---ISSEKPNGIYSIYNWHQLYHSSYY SiaTpph (145) FYDDGSAEYVRLYDFSRLPESEQEYKISLSKDNIQSSING---TQPFDNSIENIYGFSQLYPTTYH

SiaTspi (88) LIDDGSSEYQKMFYWQRLSSEEQTRDLATGLKNLKSYLSGNDNKLLRLLTGHSNKLPRRLSSFMNWHQLFPTTYH

SiaTspi-U (120) LIDDGSSEYQKMFYWQRLSSEEQTRDLATGLKNLKSYLSGNDNKLLRLLTGHSNKLPRRLSSFMNWHQLFPTTYH

301 375 SiaTple (288) FLRKDYLTVETKLHDLREYLGGSLKQMSWDTFSQ--LSKGDKELFLNIVGFDQEKLQQEYQQSE-LPNFVFTGTT SiaTpph (208) MLRADIFETNLPLTSLKRVISNNIKQMKWDYFTT--FNSQQKNKFYNFTGFNPEKIKEQYKASP-HENFIFIGTN SiaTspi (163) MLRMDYLDK-PELHQLKQYLGNNAQQIRWNYIADNLFDDEQQSLFYQLLGISLAEQKQLRAGRQQLHDFMFIGVD

SiaTspi-U (195) MLRMDYLDK-PELHQLKQYLGNNAQQIRWNYIADNLFDDEQQSLFYQLLGISLAEQKQLRAGRQQLHDFMFIGVD

376 450 SiaTple (360) TWAGGETKEYYAQQQVNVVNNAINETSP--YYLGREHDLFFKGHPRGGIINDIILGSFNNMIDIPAKVSFEVLMM

SiaTpph (280) SGT---ATAEQQIDILTEAKKPDSPIITNSIQGLDLFFKGHPSATYNQQIID--AHNMIEIYNKIPFEALIM

SiaTspi (237) SSN---ASSKLQINVIADSRQESG--IIPTITAKKMLFKGHPFANFNQTIVD--AHQMGEMPAMIPFETLIM

SiaTspi-U (269) SSN---ASSKLQINVIADSRQESG--IIPTITAKKMLFKGHPFANFNQTIVD--AHQMGEMPAMIPFETLIM

451 520 SiaTple (433) TGMLPDTVGGIASSLYFSIPAEKVSFIVFTSSDTITDREDALKSPLVQVMMTLGIVKEKDVLFWC--- SiaTpph (347) TDALPDAVGGMGSSVFFSLPNTVENKFIFYK---SDTDIENNALIQVMIELNIVNRNDVKLISDLQ-- SiaTspi (302) TGNLPQKVGGMASSLYFSLPNNYHIEYIVFSG----SKKDLEQHALLQIMLYLKVISPERVYFSEQFKSC

SiaTspi-U (334) TGNLPQKVGGMASSLYFSLPNNYHIEYIVFSG----SKKDLEQHALLQIMLYLKVISPERVYFSEQFKSC

Sequence 11.1. Protein sequence alignment of SiaTspi and SiaTspi-U with 2,3SiaTpph and 2,6SiaTple. Red frames

indicate upstream sequence and 12 amino acid insertion in the middle of SiaTspi.

However, an interesting observation is that the gene of SiaTspi listed in the Genbank and the

CAZy database contains only 1,101 bp encoding a predicted protein of 367 amino acid residues, which is significantly shorter than other SiaTs in the same family (usually around 400 amino acid residues for 2,3SiaT and 500 amino acid residues for 2,6SiaT), while this

might be the result of evolution to adapt the organism to its preferred cold and pressurized habitat, it could also be due to a mistake generated by the automated sequence analysis software which might have misinterpreted the start codon ATG of the open reading frame. Using BLAST, indeed a missed sequence fragment of 96 bp can be identified. This sequence fragment codes for an additional series of amino acid residues that shows high similarity with the corresponding part of other SiaTs (Sequence 11.1, red frame 1). When this sequence is joined to the SiaTspi, SiaTspi reaches the full sequence length typical for other 2,3SiaTs. Thus, it

is inferred that this sequence might be an essential functional part of SiaTspi. On the other

hand, a corresponding 24 amino acid sequence at the N-terminus of 2,3SiaTpph has been

identified as a common signal peptide sequence, which does not contribute to the sialyltransfer function of the enzyme.[161]

As an organism living in the deepsea, S. piezotolerans has to adapt its metabolism and constituents to the cold and pressurized environment. Therefore, the SiaTspi might be likely a

cold-adapted enzyme. Normally, cold-adapted enzymes have remarkable sequence signatures. For example, they contain lower numbers of polar (Ser, Thr, Asn, Glu and so on) and aromatic (Tyr, Phe and Trp) amino acid residues to reduce the number of stabilizing hydrogen bonds and aromatic π- and hydrophobic interactions.[227, 228] The number of Pro residues is also reduced because of the rigidity of its imino group.[227] Gly stacking may be found near the active site because its absent side chain offers varied dihedral angle in protein conformation, which is beneficial for the flexibility of the enzyme.[227] Because the guanidinium group of Arg which can form at least one salt bridge in the tertiary structure, a lower number of Arg residues or a reduced ratio of Arg/(Arg+Lys) is often can be observed in thermolabile enzymes.[227]

To find out whether SiaTspi fits the features of a cold-adapted enzyme, its amino acid residue

composition and secondary structure were analyzed using the 2,3SiaTpph from a mesophilic

source as reference. The analysis data is listed in Table 11.1. Compared to 2,3SiaTpph, the

percentage of polar amino acid residues is obviously reduced. On the other hand, the total number of aromatic residues is almost the same as in 2,3SiaTpph, and both of them contain the

same number of Pro residues. The Gly content in SiaTspi is slightly higher than that found in

2,3SiaTpph. All these data show that SiaTspi may have a lower thermostability than 2,3SiaTpph.

However, the doubled number of Arg and ratio Arg/(Lys+Arg) suggests the opposite conclusion. Considering the relative low optimum temperature of 2,3SiaTpph (30°C),[161] the

analysis for which partly fits the property of a cold-adapted enzyme itself,[229] the SiaTspi

Table 11.1. Amino acid residue composition and secondary structure analysis of SiaTspi and 2,3SiaTpph

SiaTspi SiaTspi-U dSiaTspi-U 2,3SiaTpph

Total residues 367 399 387 409 Ser 9.68% 9.52% 9.82% 8.31% Thr 3.72% 3.76% 3.26% 6.36% Asn 5.71% 5.76% 5.43% 10.02% Glu 4.42% 4.26% 4.39% 6.11% Tyr 4.47% 4.51% 4.65% 3.64% Phe 3.97% 4.01% 4.13% 6.36% Trp 1.24% 1.25% 1.29% 0.73% Pro 3.54% 3.76% 3.88% 3.67% Gly 5.18% 4.76% 4.65% 4.16% Arg 3.72% 3.76% 3.62% 1.96% Arg/(Lys+Arg) 41.7% 40.5% 40.0% 20.5%

Disulfide linkage none none none none