The presence of multiple transmembrane domains has hampered biochemical studies of MBOAT acyltransferases in general, and Hhat in particular. With the exception of two residues that have been shown to be required for enzymatic activity, there has been no structure-function analysis of Hhat. In this study, we identify specific regions and multiple residues within Hhat that regulate protein stability and/or catalysis. Of note, alignment of the sequences of MBOAT proteins that acylate protein substrates revealed the presence of an additional region of high sequence conservation () that had not been previously identified. Here we report the results of our analyses of 10 deletion mutants and 11 point mutants within Hhat.
Many of the mutants exhibited increased rates of protein degradation compared to WT Hhat, and nearly all of the mutants in this class had defects in Shh palmitoylation activity. This was particularly evident when truncations were made at the N- or C-terminus of Hhat. However, steady state levels of these mutants, as detected by anti-HA Western blotting in the absence of cycloheximide, appeared to be similar to that of WT Hhat. We quantified the rate of synthesis of Hhat using Tran-35S-labeling, and found no change in the rate of synthesis of the mutants compared to WT Hhat (data not shown). One possible explanation to reconcile the observed differences in stability is to postulate that ongoing protein synthesis is required to maintain mutant Hhat protein levels. If the Hhat truncation mutants are misfolded, they could be present at equivalent levels to WT Hhat but would likely be more susceptible to degradation, especially when protein levels are not replenished (ie in the presence of cycloheximide). In this case, misfolding of the mutant proteins might account for the decreased Hhat activity. Alternatively, the truncation mutants could be inactive because regions involved in substrate recognition or catalysis were deleted.
Most of the internal deletion and point mutants were as or nearly as stable as WT Hhat but had reduced PAT activity. In Hhat, these include deletions of residues 187–192, 228–234, and 368–380, as well as the point mutants F338A, D339A, W378A, and H379A. The equivalents of residues F338 and D339 are moderately conserved in the MBOAT family (FD in Hhat and GUP1, FN in LPAT5, and WN in the other family members). W378 and H379 are present in all MBOAT family members, except for GUP1 (Leu in place of His) and Porc and ACAT1 and 2 (Leu or Val in place of Trp). Mutations of residues corresponding to H379A or W378A have been reported in both LPAT and ACAT family members. Mutating either residue abolishes activity in all LPAT family members tested 
. The conserved His is also absolutely required for ACAT activity 
. However, mutation of the Val residue at the position corresponding to W378 compromises not only enzymatic activity but also protein expression, complicating its analysis 
In addition to the canonical MBOAT homology domain, we also identified residues in a second region (residues 196– 234) that are highly conserved in family members that transfer fatty acids onto protein substrates. Of these, the Tyr at position 207 is also conserved in both LPAT and ACAT family members, whereas the Gly at position 217 is conserved among LPAT but not ACAT family members. In ACAT family members the residue at this position is either Ala or Cys. To date there are no other reports of mutation within this region of another MBOAT family member. It will be interesting to see if residues within this region are important specifically for transfer of fatty acids onto proteins or if they are more broadly required for activity within the MBOAT family.
A prior study reported that an Hhat construct with both D339 and H379 mutated to Ala was not able to rescue the phenotype of an Hhat-defective mutant 
. We have analyzed the effects of each of these mutations separately. D339A Hhat was essentially inactive (<7% of WT activity). H379 has been proposed to be part of the active site of MBOAT proteins. Mutation of this conserved Histidine residue completely abrogates activity for all tested members of the MBOAT family leading to the stipulation that it is directly involved in catalysis. However, the H379A mutant retains 50% of the activity of WT Hhat, suggesting that this residue is not absolutely required for catalysis. Kinetic analyses performed on purified H379A Hhat revealed that this mutant binds palmitoyl CoA with an affinity similar to WT Hhat (). This suggests that H379 may be more important for recognition and binding of Shh. Mutation of the adjacent residue, W378, caused a more severe effect on Hhat activity. W378A Hhat exhibited alterations in apparent Km
for both Shh and Iodopalmitoyl CoA substrates. Given the effect on both parameters it is not clear whether the W378A mutant is compromised in catalytic activity, has a severe defect in substrate binding, or a combination of the two. Direct measurements of substrate binding will be required to determine which is the case.
One of the hallmarks of the palmitoylation reaction catalyzed by the other family of PATs, DHHC PATs, is their ability to autoacylate 
. By contrast, we have not detected acyl-enzyme formation for Hhat and palmitate. Co-immunoprecipitation and pulldown assays aimed at monitoring Hhat interactions with Shh and palmitoylCoA were performed using full length Shh, recombinant ShhN purified from E.coli
, as well as a biotinylated Shh peptide that we have previously shown acts as a Hhat substrate in vitro 
. We were unable to detect stable or specific interactions of Hhat with any of these substrates. This is not surprising given the hydrophobic nature of the players involved and the fact that enzymes are not expected to bind with high affinity to their substrates as this would tend to hinder enzymatic turnover. Thus, we have not been able to utilize direct binding assays to quantify the interactions between Hhat and its two substrates. Instead, we purified two Hhat mutants, W378A and H379A, to apparent homogeneity, and showed that these mutants exhibited kinetic alterations that may explain their catalytic defects as described above.