CrtW structure and function
5.2. The secondary structure of CrtW
CrtW is predicted to play a role in the process of light-induced carotenogenesis, and as a result is most likely to be located at the cell membrane. This is where the majority of carotenoid biosynthetic enzymes are already known to be located. This suggestion is supported by TMpred, which predicts that the protein possesses a number of transmembrane regions. Two potential helical arrangements were suggested – Model 1 and Model 2, with the most likely of the two being Model 1 due to its higher score (Table 5.1). For Model 1 each of five helices span the
membrane, and the protein has an N-terminal extension located inside the cell and a C-terminal extension located outside of the cell membrane. In terms of helix length, each is similar in size, although the overall scores for each do vary greatly, ranging from one at 2177 to another at just 870. Model 2 is constructed of four helices, each of more varying size than in Model 1. In this instance though, the individual helix scores are much closer together in value and the final predicted protein would have both the N-terminal and C-terminal extensions on the inner surface of the cell membrane (Table 5.1).
Model 1 Helix length (a.a.) Score Orientation (in cell)
1 22 2177 Inside – Outside 2 19 870 Outside – Inside 3 21 1008 Inside – Outside 4 20 1022 Outside – Inside 5 20 1477 Inside – Outside Model 2 1 22 1865 Inside – Outside 2 18 1495 Outside – Inside 3 24 1194 Inside – Outside 4 20 1477 Outside - Inside
Table 5.1A table depicting the two predicted helical arrangements of CrtW by TMpred.
This is not the first time that the protein structure has been modelled of a
member of the β-ketolase family. A study was recently conducted of the CrtW
produced by the α-proteobacterium Paracoccus sp. Strain N81106 (Yeet al., 2006).
Part of the study included initial prediction of the secondary protein structure, which determined that four transmembrane helices were encoded by the amino acid sequence. This is the same as predicted in Model 2 for M. xanthus CrtW, which leaves both termini on the inside of the cell. Using this precedent it appears most likely that Model 2 is correct, rather than Model 1. Also in the same study a direct sequence alignment was carried out comparing the Paracoccus sp. Strain N81106 protein sequence with those ofBrevundimonas sp. SD212 (α-proteobacterium) and an Alcaligenes species (β-proteobacterium). This was done in an attempt to identify any similarities that existed between the three distantly related organisms and to determine whether there were other additional key elements encoded, as predicted from the amino acid sequence. For the purpose of this study the alignment was repeated, but also incorporating recent sequence data from Myxococcus xanthus (δ- proteobacterium) DK1622, Algoriphagus species (Bacteroidetes/Chlorobi group) and
Bradyrhizobium (α-proteobacterium) ORS278 (Figure 5.1). Key features were found to be highly conserved in all of the proteins.
One of the features that was identified during the original comparison between the three species was the presence of three histidine-rich (His) motifs which were conserved in each CrtW protein (Ye et al., 2006). These His features were spread along the peptide chain, but it was predicted each was still located on the inner surface of the membrane. Two of the motifs are located between the second and third helices, and the other immediately following the fourth helix at the C-terminus. Further analysis of each individual motif, whereby a selection of the histidine residues were mutated, established that the majority were essential for effective CrtW ketolase activity in Paracoccus species. In any instances where enzymatic activity was still observed following mutation, it was always found to be greatly reduced. Identical His motifs were present in each species, includingM. xanthus (Figure 5.1). They are all highly conserved, implying their importance, although the second histidine residue in His3 is substituted with an aromatic residue in both M. xanthus and Algoriphagus species. Mutation studies of this residue in Paracoccus species resulted in reduced overall levels of substrate interaction, but the motif itself still remained functional (Ye et al., 2006). All of the identified His motifs were previously proven to be involved in iron coordination, enabling the enzyme access to electron donors.
In addition to the three His motifs, a further conserved amino acid sequence was located between His2 and the third transmembrane helix. This motif was conserved in all of the six species and consists of DPDF. The motif is believed to play a similar role in aiding iron coordination to that of the His motifs. In addition there is a separate histidine residue located immediately after the final helix, which although not forming part of a larger motif may still play a significant role during catalysis. This is supported by the fact that in its absence ketolase activity is greatly reduced. Mutation of a single phenylalanine (F) amino acid residue in theParacoccus sequence reduced ketolase activity (Yeet al., 2006). This residue was found to form part of a far larger motif encoded by the sequence W(xxx)F(xxx)Y (wherexrepresents any amino acid residue). Once again it was a highly conserved protein region present in each of the six CrtW peptides and was predicted to be found on the cytosolic side of the cell membrane (Figure 5.1).
Bradyrhizobium sp. ---MHAATAKATEFGASRRDDARQRRVGLTLAAVIIAAWLV 38
Brevundimonas sp. ---MTAAVAEP---RIVPRQTWIGLTLAGMIVAGWGS 31
Paracoccus sp. ---MSAHALPK---ADLTATSLIVSGGIIAAWLA 28
Alcaligenes sp. ---MSGRKPGT---TGDTIVNLGLTAAILLCWLV 28
Myxococcus sp. ---METSARQLR---PAPPGPWGVVIALIIMGAWGG 30
Algoriphagus sp. MADGGSEGKDSDFLRKHSQLAEMKAEIT---SMSVDPKGIFIAVAIIGLWFS 49
¯ ¯
<--- Helix 2 ---> XXXXX Bradyrhizobium sp. LHVGLMFFWPLTLHSLLPALPLVVLQTWLYVGLFIIAHDCMHGSLVPFKPQVNRRIGQLC 98
Brevundimonas sp. LHVYGVYFHRWGTSSLVIVPAIVAVQTWLSVGLFIVAHDAMHGSLAPGRPRLNAAVGRLT 91
Paracoccus sp. LHVHALWFLDAAAHPILAIANFLGL-TWLSVGLFIIAHDAMHGSVVPGRPRANAAMGQLV 87
Alcaligenes sp. LHAFTLWLLDAAAHPLLAVLCLAGL-TWLSVGLFIIAHDAMHGSVVPGRPRANAAIGQLA 87
Myxococcus sp. HLAWALTRAELPWVEPLTWLHVALQ-AWLCTGLFITGHDAMHG-TVSGRRWVNEAVGTVA 88
Algoriphagus sp. SLVFLLN-YEISWSDPLVYLGILVQ-MHLYTGLFITAHDAMHG-LVASNKRLNTSIGWVS 106
¯¯¯¯¯ ¯ ¯
His1
XXXXXXXXX <--- Helix 3 -- Bradyrhizobium sp. LFLYAGFSFDALNVEHHKHHRHPGTAEDPDFDEVPPHGFWHWFASFFLHYFGWKQVAIIA 158
Brevundimonas sp. LGLYAGFRFDRLKTAHHAHHAAPGTADDPDFYAPAPRAFLPWFLNFFRTYFGWREMAVLT 151
Paracoccus sp. LWLYAGFSWRKMIVKHMAHHRHAGTDDDPDFDHGGP---VRWYARFIGTYFGWREGLLLP 144
Alcaligenes sp. LWLYAGFSWPKLIAKHMTHHRHAGTDNDPDFGHGGP---VRWYGSFVSTYFGWREGLLLP 144
Myxococcus sp. CFLFAGLSYRRLVVNHRAHHARPTSDADPDFSTHSQS-FWPWLGTFMARYTTLPQLGVMA 147
Algoriphagus sp. ALLFSYNFYSKLFPKHHEHHRFVATDQDPDFHT-SDN-FFVWYFSFIKQYITLWQIILMA 164
¯¯¯¯¯ ¯¯¯¯ ¯ ¯ ¯
His2 DPDF WxxxFxxxY
---> <--- Helix 4 --->
Bradyrhizobium sp. AVSLVYQLVFAVPLQNILLFWALPGLLSALQLFTFGTYLPHKPATQPFADRHNARTSEFP 218
Brevundimonas sp. ALVLIALFGLGARPANLLTFWAAPALLSALQLFTFGTWLPHRHTDQPFADAHHARSSGYG 211
Paracoccus sp. VIVTVYALILGDRW-MYVVFWPLPSILASIQLFVFGTWLPHRPGHDAFPDRHNARSSRIS 203
Alcaligenes sp. VIVTTYALILGDRW-MYVIFWPVPAVLASIQIFVFGTWLPHRPGHDDFPDRHNARSTGIG 203
Myxococcus sp. AKFNVL-LFLGVSQPHILGYWVLPSVLGTLQLFYFGTYLPHRRPETPDMAPHHARTLPRN 206
Algoriphagus sp. ITFNVLKLFLPVD--NLIIFWMLPAVLSTFQLFYFGTYLPHKGVND---NKHHSTTQSKN 219
¯¯ ¯¯ ¯¯ ¯
H
XXXXXXXX
Bradyrhizobium sp. AWLSLLTCFHFG-FHHEHHLHPDAPWWRLPEIKRRALERRD--- 258
Brevundimonas sp. PVLSLLTCFHFG-RHHEHHLTPWRPWWRLWRGES--- 244
Paracoccus sp. DPVSLLTCFHFGGYHHEHHLHPTVPWWRLPSTRTKGDTA--- 242
Alcaligenes sp. DPLSLLTCFHFGGYHHEHHLHPHVPWWRLPRTRKTGGRA--- 242
Myxococcus sp. HLWALLSCFFFG-YHWEHHESPGTPWWRLWRLKDARAREAALTQSTGTLPGQEGTAR 262
Algoriphagus sp. HFSAFITCYFFG-YHYEHHDSPGTPWWRLWRVKESQSN--- 256
¯ ¯ ¯¯¯¯¯ ¯ ¯¯ ¯ His3
Black shading Identical residues Dark grey shading Conserved substitutions Pale grey shading Semi-conserved substitutions
Figure 5.1A ClustalW alignment of CrtW sequences for six bacterial species;Bradyrhizobiumsp. ORS278 (GenBank accession number AAF78203), Brevundimonas sp. SD212 (GenBank accession number AB181388),Paracoccussp. N81106 (GenBank accession number ABL09497),Alcaligenessp. (NCBI accession number BAA09596), Myxococcus xanthus DK1622 (GenBank accession number ABF90074) and Algoriphagus sp. (GenBank accession number ABB88952). Any unlabelled underlined bases are previously identified from a comparative CrtW study as being highly conserved (Yeet al., 2006). A string ofXs indicate the area in which hydrophobic regions have been identified. The conserved motifs His1, His2, His3, DPDF, H, and WxxxFxxxY are marked and are believed to be involved in metal coordination (See text).
Helix domain 1: Amino acids 17-30 Helix domain 2: Amino acids 44-66 Helix domain 3: Amino acids 137-156 Helix domain 4: Amino acids 162-184
Figure 5.2Two topographic models of the predicted secondary structure for CrtW fromM. xanthus. The helices are coloured pale green (1-4), His motifs lime green (H1-3), the hydrophobic regions a deep sea green and conserved motifs labelled yellow. The difference in appearance is dependent upon whether the hydrophobic regions interact with each other or the membrane.
H1 H2
1
2
3
NH2
COOH
CYTOSOL OUTSIDE CELL Inner leaflet Outer leaflet H34
H DPDF WxxxFxxxY H11
2
4
NH2
COOH
CYTOSOL OUTSIDE CELL Inner leaflet Outer leaflet H33
H DPDF WxxxFxxxY H2The final elements identified during the initial ketolase modelling process were two highly hydrophobic regions. These consisted of various hydrophobic residues, but all were located in the same regions in each CrtW (highlighted in Figure 5.1). Simplified versions of the predicted CrtW topographic model can be seen in Figure 5.2. The difference between the two models is whether the hydrophobic regions interact with the membrane or each other. To fully understand the role CrtW plays in M. xanthus carotenogenesis, experimental evidence was still required. In terms of carotenoid production the preferred method of study is usually through the creation of a gene knockout strain and examination of the resultant phenotype.